Compositions and methods for antibody production

ABSTRACT

Compositions and methods for minimizing antibody disulfide bond reduction are described. In one aspect, a composition is provided for culturing mammalian host cells to express an antibody including an anti-reduction agent that minimizes reduction of a disulfide bond in the antibody or fragment thereof. In some other aspects, methods for minimizing disulfide bond reduction; increasing production of an antibody or fragment thereof with intact native disulfide bonds; increasing a ratio of non-reduced to reduced antibody or fragment thereof; producing a therapeutic antibody or fragment thereof by adding a sufficient amount of an anti-reduction agent to a cell culture media, pre-harvest cell culture fluid, or harvest cell culture fluid are described. In another aspect, minimizing disulfide bond reduction in an antibody or fragment thereof culturing the host cell in a concentration of at least about 20% O 2  is described.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.62/507,528, filed May 17, 2017. The entire contents of that applicationare hereby incorporated herein.

BACKGROUND OF THE INVENTION

Antibodies, or immunoglobulins, contain heavy and light chains that areheld together by non-covalent interactions as well as by covalentinterchain disulfide bonds. Human immunoglobulin G (IgG) isotypes, IgG1,IgG2, IgG3, and IgG4, contain one disulfide bond between the heavy chainand light chain, whereas the number of disulfide bonds between the twoheavy chains is two for IgG1 and IgG4, four for IgG2, and eleven forIgG3. The inter-chain disulfide bonds in the antibody are moreaccessible to solvent than intrachain disulfide bonds, and can bereduced to thiol residues by dithiol agents, such as dithiothreitol.

Several monoclonal antibody based therapeutics, which include theantibody-drug conjugates brentuximab vedotin and ado-trastuzumabemtansine, are currently approved for clinical use in variousindications, such as oncology and rheumatoid arthritis. Theserecombinant monoclonal antibodies are produced at high titers in cells,such as CHO, SP2/0, and NS0 cells. The recombinant antibodies aregenerated by mammalian cells that secrete the antibodies into themedium. At the end of the antibody production process, the cells areseparated from the antibody-containing medium using methods, such astangential flow micro filtration, centrifugation, depth filtration,flocculation or precipitation and then purified, for example, byaffinity chromatography. During the cell separation step, cell damagemay occur causing the release of intracellular reducing proteins.Without wishing to be bound by theory, the release of such proteinscould undesirably reduce the inter-chain disulfide bonds present inantibodies or other recombinant proteins.

The cysteine content of mammalian proteins is typically about twopercent, of which about 70% cysteine thiols are exposed and availablefor redox reactions. This suggests that a large number of intracellularproteins could be involved in intracellular redox homeostasis. In onestudy, 24 thiol proteins sensitive to oxidation were identified in ahuman cell line, including glyceraldehyde-3-phosphate dehydrogenase,peroxyredoxin 2, glutathione-S-transferase P1-1, enolase, Protein kinaseA subunit, annexin VI, serine/threonine kinase BUB1β, heat-shock protein90β, and proteosome components. Other studies have reported theanti-oxidant functions of thioredoxin and glutaredoxin systems in cells.A significantly high percentage (5%) of soluble cellular proteins havevicinal thiol groups, which could have high reduction potential. About5-15% of mitochondrial proteins are reported to have vicinal thiolgroups. All of these thiol-containing intracellular proteins could bereleased by cell damage during the cell separation step prior to theantibody purification step, and could lead to the reduction of theantibody inter-chain disulfide groups. Given that the native,inter-chain disulfide bonds in the antibody contribute to thethermodynamic stability of the antibody, any reduction could lead toinstability of the antibody, which is undesirable in a therapeuticantibody or antibody drug conjugate. Improved methods for protectingdisulfide bonds present in antibodies and/or other recombinant proteinsare needed.

SUMMARY OF THE INVENTION

As described below, the invention features compositions and methods forminimizing fragmentation and disulfide bond reduction in antibodies andrecombinant proteins.

In one aspect, the invention provides a cell culture, harvest orpre-harvest composition (e.g., cell culture media, harvest cell culturefluid, or pre-harvest cell culture fluid) containing an effective amountof an anti-reduction agent that is any one or more of methylene blue, aquinone (e.g., a substituted benzoquinone; 1,2-naphthoquinone-4-sulfonicacid; and anthraquinone-2-sulfonic acid); a coenzyme Q analog (e.g.,coenzyme Q0 and/or coenzyme Q2), a disulfide (e.g., disulfiram; lipoicacid; a soluble cystine analog); a combination of glutathione reductaseand oxidized glutathione (GSSG); oxidized glutathione alkyl esters(e.g., oxidized glutathione methyl esters; oxidized glutathione ethylesters; oxidized glutathione isopropyl esters);5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), a salt thereof and anycombinations thereof.

In one embodiment, the substituted benzoquinone is represented byformula (I):

where R₁, R₂, R₃, and R₄ is each independently selected from the groupconsisting of H, alkyl, alkoxy, COOH, and SO₃H.

In one embodiment, the cystine analog is any one or more of cystinedimethyl ester, cystine diethyl ester, cystine methyl ester, cystineethyl ester, di-N-acetyl cystine, cystine bis(t-butyl ester), cystinemono(t-butyl ester), monoesters of cystine, asymmetric (i.e., mixed)esters of cystine, and combinations thereof. In another embodiment, thecomposition contains one or more of a mixture ofanthraquinone-2-sulfonic acid and cystine dimethyl ester; a mixture oflipoic acid and anthraquinone-2-sulfonic acid; and a mixture of lipoicacid and cystine dimethyl ester.

In another aspect, the invention provides a cell culture, harvest orpre-harvest composition (e.g., cell culture media, harvest cell culturefluid, or pre-harvest cell culture fluid) containing an effective amountof one or more of methylene blue; a substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; anthraquinone-2-sulfonic acid;lipoic acid; disulfiram; a soluble cystine analog; a combination ofglutathione reductase and oxidized glutathione (GSSG); oxidizedglutathione alkyl esters (e.g., oxidized glutathione methyl esters;oxidized glutathione ethyl esters; oxidized glutathione isopropylesters); and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB).

In yet another aspect, the invention provides a method for minimizingdisulfide bond reduction in a recombinant protein, antibody or fragmentthereof expressed in a host cell, the method involving adding ananti-reduction agent to a cell culture media, pre-harvest culture fluid,or harvest culture fluid, containing the antibody or fragment thereof,where the anti-reduction agent is any one or more of methylene blue, aquinone, a disulfide, a salt thereof and any combinations thereof.

In yet another aspect, the invention provides a method of increasing aratio of non-reduced to reduced protein, antibody, or fragment thereof,that is produced by a mammalian host cell, the method involving adding asufficient amount of an anti-reduction agent to a cell culture media,pre-harvest cell culture fluid, or harvest cell culture fluid,containing the antibody or fragment thereof, where the anti-reductionagent is any one or more of methylene blue, a quinone, a disulfide, asalt thereof, and combinations thereof.

In yet another aspect, the invention provides a method for preventing orminimizing disulfide bond reduction or fragmentation in an antibody,antibody fragment, or recombinantly expressed protein, the methodinvolving contacting the protein with an anti-reduction agent that isany one or more selected from the group consisting of methylene blue; asubstituted benzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; lipoic acid; disulfiram; a solublecystine analog; a combination of glutathione reductase and oxidizedglutathione (GSSG); oxidized glutathione alkyl esters; and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), where the antibody, antibodyfragment, or recombinantly expressed protein is contacted duringexpression in a host cell, during cell culture, pre-harvest, or harvest.

In still another aspect, the invention provides a method of increasingproduction of a protein, antibody or fragment thereof with intact nativedisulfide bonds that is expressed in a mammalian host cell, the methodinvolving adding an effective amount of an anti-reduction agent to acell culture media, pre-harvest culture fluid, or harvest culture fluid,containing the antibody or fragment thereof, where the anti-reductionagent is any one or more selected from the group consisting of methyleneblue, a quinone, a disulfide, a salt thereof, and combinations thereof.

In still another aspect, the invention provides a method of producing atherapeutic antibody, or fragment thereof, the method involving exposinga mammalian host cell that produces the therapeutic antibody or fragmentthereof, to a composition containing a sufficient amount of ananti-reduction agent in a cell culture media, pre-harvest cell culturefluid, or harvest cell culture fluid, where the anti-reduction agent isany one or more selected from the group consisting of methylene blue, aquinone, a disulfide, a salt thereof, and any combinations thereof.

In still another aspect, the invention provides a method of minimizingdisulfide bond reduction in a recombinant protein, antibody or fragmentthereof that is expressed in a mammalian host cell, the method involvingsparging the cell culture medium, pre-harvest cell culture fluid, orharvest cell culture fluid with oxygen to a concentration of at leastabout 20% dissolved O₂. In one embodiment, the concentration ofdissolved O₂ is in a range of about 20% to about 100%.

A method of minimizing disulfide bond reduction in a recombinantprotein, antibody or fragment thereof that is expressed in a mammalianhost cell, the method comprising sparging the cell culture medium,pre-harvest cell culture fluid, or harvest cell culture fluid with acombination of air and oxygen to a concentration of at least about 20%dissolved O₂. In one embodiment, the concentration of dissolved O₂ is ina range of about 20% to about 100%.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the anti-reduction agent does notcovalently modify the protein, antibody or fragment thereof. In otherembodiments of the above aspects or any other aspect of the invention,the anti-reduction agent is at a sub-stoichiometric concentration tothat of total thiol in the solution. In other embodiments of the aboveaspects or any other aspect of the invention, the anti-reduction agentis present at a concentration from about 0.01 mM to about 100 mM (e.g.,0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100);from about 0.1 mM to about 10 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In other embodiments ofthe above aspects or any other aspect of the invention, the compositionis cell culture media, harvest cell culture fluid, or pre-harvest cellculture fluid. In other embodiments of the above aspects or any otheraspect of the invention, the protein, antibody, or fragment thereof, hasa thiol:antibody ratio of at least about 25, 50, 75, 90, 95, or even100% lower in the presence of the anti-reduction agent than in theabsence of the anti-reduction agent. In other embodiments of the aboveaspects or any other aspect of the invention, the ratio is decreased byat least about 2, 5, 10, or 20-fold.

In other embodiments of the above aspects or any other aspect of theinvention delineated herein, a quinone is any one or more of asubstituted benzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; a coenzyme Q, and combinations thereof.In one embodiment, a quinone is anthraquinone-2-sulfonic acid. Inanother embodiment, the substituted benzoquinone is represented byformula (I):

where R₁, R₂, R₃, and R₄ is each independently selected from the groupconsisting of H, alkyl, alkoxy, COOH, and SO₃H.

In other embodiments of the above aspects or any other aspect of theinvention, the coenzyme Q analog is coenzyme Q0 and/or coenzyme Q2. Inother embodiments of the above aspects or any other aspect of theinvention, the disulfide is any one or more of a disulfiram; lipoicacid; a soluble cystine analog; a combination of glutathione reductaseand oxidized glutathione (GSSG); oxidized glutathione alkyl esters(e.g., oxidized glutathione methyl esters; oxidized glutathione ethylesters; oxidized glutathione isopropyl esters);5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and combinations thereof. Inone embodiment, the disulfide is lipoic acid. In other embodiments ofthe above aspects or any other aspect of the invention, the cystineanalog is any one or more of cystine dimethyl ester, cystine diethylester, cystine methyl ester, cystine ethyl ester, di-N-acetyl cystine,cystine bis(t-butyl ester), monesters of cystine, asymmetric esters ofcystine, and combinations thereof. In one embodiment, the cystine analogis cystine dimethyl ester, cystine bis(t-butyl ester), or anycombinations thereof. In another embodiment, the cystine analog iscystine bis(t-butyl ester). In yet another embodiment, the cystineanalog comprises cystine dimethyl ester (CDME) and cystine bis(t-butylester). In one preferred embodiment, the cystine bis(t-butyl ester)comprises L-cystine bis (t-butyl ester)(CDBE). In another preferredembodiment, the cystine analog is cystine dimethyl ester. In otherembodiments of the above aspects or any other aspect of the invention,the composition contains a mixture of anthraquinone-2-sulfonic acid andcystine dimethyl ester; a mixture of lipoic acid andanthraquinone-2-sulfonic acid; or a mixture of lipoic acid and cystinedimethyl ester. In other embodiments of the above aspects or any otheraspect of the invention, the method involves adding the anti-reductionagent to the cell culture medium (e.g., within about 15 minutes, 30minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 48 hoursof harvesting the cell culture). In one embodiment, the method comprisesadding the anti-reduction agent to the cell culture medium within about15 minutes of harvesting the cell culture.

In other embodiments of the above aspects or any other aspect of theinvention, the step of adding the anti-reduction agent does not decreaseviability of the cells by greater than about 15%. In other embodimentsof the above aspects or any other aspect of the invention, the methodinvolves adding the anti-reduction agent to the pre-harvest cell culturefluid or to the harvest cell culture fluid. In other embodiments of theabove aspects or any other aspect of the invention, the anti-reductionagent is added at a sub-stoichiometric concentration to that of totalthiol in the solution.

In other embodiments of the above aspects or any other aspect of theinvention, the anti-reduction agent is added at a molar ratio of about0.1 to about 0.8 of a total thiol concentration in the cell culturemedia, pre-harvest cell culture fluid, or harvest cell culture fluid. Inanother embodiment, the anti-reduction agent is added at a molar ratioof about 0.1 to about 10 of a total thiol concentration in the cellculture media, pre-harvest cell culture fluid, or harvest cell culturefluid. In other embodiments of the above aspects or any other aspect ofthe invention, the anti-reduction agent is added to a finalconcentration in a range from about 0.01 mM to about 100 mM. In otherembodiments of the above aspects or any other aspect of the invention,the final concentration of the anti-reduction agent ranges from about0.1 mM to about 10 mM. In other embodiments of the above aspects or anyother aspect of the invention, the method involves sparging the cellculture medium, pre-harvest cell culture fluid, or harvest cell culturefluid with air or oxygen to a concentration of at least about 20%dissolved O₂. In other embodiments of the above aspects or any otheraspect of the invention, the concentration of dissolved O₂ is in a rangeof about 20% to about 100% (e.g., 20, 25, 50, 75, 90, 95, 99, 100%).

In other embodiments of the above aspects or any other aspect of theinvention, the mammalian host cell is a Chinese Hamster Ovary (CHO)cell. In other embodiments of the above aspects or any other aspect ofthe invention, the antibody is any one or more of an anti-FOLR1 antibody(e.g., SEQ ID NO.: 3, 4, or 5), an anti-CD56 antibody (e.g., huN901), ananti-CD37 antibody, an anti-EGFR antibody, an anti-IGF-1R antibody, ananti-MUC1, an anti-CA6 glycotope, an anti-CD19 antibody, and ananti-CD33 antibody. In other embodiments of the above aspects or anyother aspect of the invention, the antibody is at least one of an IgG1,IgG2, IgG3, and IgG4 isotype. In one embodiment, the antibody is an IgG1isotype. In other embodiments of the above aspects or any other aspectof the invention, the antibody or fragment thereof is not covalentlymodified by the anti-reduction agent. In other embodiments of the aboveaspects or any other aspect of the invention, the method does notincrease immunogenicity of the antibody or fragment thereof. In otherembodiments of the above aspects or any other aspect of the invention,the antibody is recombinantly expressed in the host cell. In otherembodiments of the above aspects or any other aspect of the invention,the antibody is any one or more of a therapeutic antibody, a modifiedantibody and a conjugated antibody.

In another aspect, the disclosure provides a method for minimizingdisulfide bond reduction in a recombinant protein, antibody or fragmentthereof expressed in a host cell. The method comprises adding ananti-reduction agent to a cell culture media, pre-harvest cell culturefluid, or harvest cell culture fluid, comprising the antibody orfragment thereof, wherein the anti-reduction agent is selected from thegroup consisting of methylene blue, a quinone, a disulfide, a saltthereof and any combinations thereof.

In yet another aspect, the disclosure provides a method for preventingor minimizing disulfide bond reduction or fragmentation in an antibody,antibody fragment, or recombinantly expressed protein. The methodcomprises contacting the protein with an anti-reduction agent selectedfrom the group consisting of methylene blue; a substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; anthraquinone-2-sulfonic acid;lipoic acid; disulfiram; a soluble cystine analog; a combination ofglutathione reductase and oxidized glutathione (GSSG); oxidizedglutathione alkyl esters; oxidized glutathione methyl esters; oxidizedglutathione ethyl esters; oxidized glutathione isopropyl esters; and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), wherein the antibody,antibody fragment, or recombinantly expressed protein is contactedduring expression in a host cell, during cell culture, pre-harvest, orharvest.

In some embodiments of the methods disclosed herein, the protein,antibody, or fragment thereof, has a thiol:antibody ratio of at leastabout 25% lower in the presence of the anti-reduction agent than in theabsence of the anti-reduction agent.

In some embodiments of the methods disclosed herein, the protein,antibody, or fragment thereof, has a thiol:antibody ratio of at leastabout 50% lower in the presence of the anti-reduction agent than in theabsence of the anti-reduction agent.

In another aspect, the disclosure provides a method of increasingproduction of a protein, antibody or fragment thereof with intact nativedisulfide bonds that is expressed in a mammalian host cell. The methodcomprises adding an effective amount of an anti-reduction agent to acell culture media, pre-harvest cell culture fluid, or harvest cellculture fluid, comprising the antibody or fragment thereof, wherein theanti-reduction agent is selected from the group consisting of methyleneblue, a quinone, a disulfide, a salt thereof, and any combinationsthereof.

In some embodiments of the methods disclosed herein, the protein,antibody, or fragment thereof, has a thiol:antibody ratio of at leastabout 25% lower in the presence of the anti-reduction agent than in theabsence of the anti-reduction agent.

In some embodiments of the methods disclosed herein, the protein,antibody, or fragment thereof, has a thiol:antibody ratio of at leastabout 50% lower in the presence of the anti-reduction agent than in theabsence of the anti-reduction agent.

Another aspect of the disclosure provides a method of increasing a ratioof non-reduced to reduced protein, antibody, or fragment thereof, thatis produced by a mammalian host cell. The method comprises adding asufficient amount of an anti-reduction agent to a cell culture media,pre-harvest cell culture fluid, or harvest cell culture fluid,comprising the antibody or fragment thereof, wherein the anti-r agent isselected from the group consisting of methylene blue, a quinone, adisulfide, a salt thereof, and combinations thereof.

In some embodiments of the methods disclosed herein, the ratio isincreased by at least about 2-fold. In some embodiments of the methodsdisclosed herein, the ratio is increased by at least about 10-fold.

Another aspect of the disclosure provides a method of producing atherapeutic antibody, or fragment thereof. The method comprises exposinga mammalian host cell that produces the therapeutic antibody or fragmentthereof, to a composition comprising a sufficient amount of ananti-reduction agent in a cell culture media, pre-harvest cell culturefluid, or harvest cell culture fluid, wherein the anti-reduction agentis at least one selected from the group consisting of methylene blue, aquinone, a disulfide, a salt thereof, and any combinations thereof.

In some embodiments, the quinone is selected from the group consistingof a substituted benzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; a coenzyme Q, and combinations thereof.In certain embodiments, the quinone is anthraquinone-2-sulfonic acid. Insome embodiments, the substituted benzoquinone is represented by formula(I):

wherein R1, R2, R3, and R4 is each independently selected from the groupconsisting of H, alkyl, alkoxy, COOH, and SO3H.

In some embodiments of the methods disclosed herein, the coenzyme Qanalog is selected from the group consisting of coenzyme Q0, coenzymeQ2, and combinations thereof.

In some embodiments of the methods disclosed herein, the disulfide isselected from the group consisting of a disulfiram; lipoic acid; asoluble cystine analog; a combination of glutathione reductase andoxidized glutathione (GSSG); oxidized glutathione alkyl esters;

oxidized glutathione methyl esters; oxidized glutathione ethyl esters;oxidized glutathione isopropyl esters; 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB) and any combinations thereof. In some embodiments, thedisulfide is lipoic acid.

In some embodiments of the methods disclosed herein, the cystine analogis selected from the group consisting of cystine dimethyl ester, cystinediethyl ester, cystine methyl ester, cystine ethyl ester, di-N-acetylcystine, L-cystine bis(methyl ester), L-cystine bis(t-butyl ester), andcombinations thereof. In some embodiments, the cystine analog is cystinedimethyl ester, L-cystine bis(t-butyl ester), or combinations thereof.

In some embodiments of the methods disclosed herein, the compositioncomprises one or more selected from the group consisting of a mixture ofanthraquinone-2-sulfonic acid and cystine dimethyl ester; a mixture ofanthraquinone-2-sulfonic acid and cystine bis(t-butyl) ester; a mixtureof lipoic acid and anthraquinone-2-sulfonic acid; a mixture of lipoicacid and cystine dimethyl ester; a mixture of lipoic acid and cystinebis(t-butyl) ester, or any combinations thereof.

In some embodiments of the methods disclosed herein, the methodcomprises adding the anti-reduction agent to the cell culture medium. Insome embodiments, the method comprises adding the anti-reduction agentto the cell culture medium within about 48 hours of harvesting the cellculture. In some embodiments, the method comprises adding theanti-reduction agent to the cell culture medium within about 24 hours ofharvesting the cell culture. In still other embodiments, the methodcomprises adding the anti-reduction agent to the cell culture mediumwithin about 12 hours of harvesting the cell culture. In furtherembodiments, the method comprises adding the anti-reduction agent to thecell culture medium within about 15 minutes of harvesting the cellculture.

In some embodiments of the methods disclosed herein, the step of addingthe anti-reduction agent does not decrease viability of the cells bygreater than about 15%.

In some embodiments of the methods disclosed herein, the methodcomprises adding the anti-reduction agent to the pre-harvest cellculture fluid.

In some embodiments of the methods disclosed herein, the methodcomprises adding the anti-reduction agent to the harvest cell culturefluid.

In some embodiments of the methods disclosed herein, the anti-reductionagent is added at a sub-stoichiometric concentration.

In some embodiments of the methods disclosed herein, the anti-reductionagent is added at a molar ratio of about 0.1 to about 10 of a totalthiol concentration in the cell culture media, pre-harvest cell culturefluid, or harvest cell culture fluid. In some embodiments, theanti-reduction agent is added to a final concentration in a range fromabout 0.01 mM to about 100 mM. In further embodiments, the finalconcentration of the anti-reduction agent ranges from about 0.1 mM toabout 10 mM.

In some embodiments of the methods disclosed herein, the methodcomprises sparging the cell culture medium, pre-harvest cell culturefluid, or harvest cell culture fluid with air or oxygen to aconcentration of at least about 20% dissolved 02. In some embodiments,the concentration of dissolved 02 is in a range of about 20% to about100%.

Yet another aspect of this disclosure provides a method of minimizingdisulfide bond reduction in a recombinant protein, antibody or fragmentthereof that is expressed in a mammalian host cell. The method comprisessparging the cell culture medium, pre-harvest cell culture fluid, orharvest cell culture fluid with oxygen to a concentration of at leastabout 20% dissolved O₂.

One aspect of the disclosure provides a method of minimizing disulfidebond reduction in a recombinant protein, antibody or fragment thereofthat is expressed in a mammalian host cell. The method comprisessparging the cell culture medium, pre-harvest cell culture fluid, orharvest cell culture fluid with a combination of air and oxygen to aconcentration of at least about 20% dissolved 02.

In some embodiments of the methods disclosed herein, the concentrationof dissolved 02 is in a range of about 20% to about 100%.

In some embodiments of the methods disclosed herein, the mammalian hostcell is a Chinese Hamster Ovary (CHO) cell.

In some embodiments of the methods disclosed herein, the antibody isselected from an anti-FOLR1 antibody, an anti-CD56 antibody, ananti-CD37 antibody, an anti-EGFR antibody, an anti-IGF-1R antibody, ananti-MUC1, an anti-CA6 glycotope, an anti-CD19 antibody, and ananti-CD33 antibody. In some embodiments, the anti-FOLR1 antibodycomprises a heavy chain or light chain variable region sequencerepresented by SEQ ID NO.: 3, 4, or 5. In some embodiments, theanti-CD56 antibody is huN901.

In some embodiments of the methods disclosed herein, the antibody is atleast one of an IgG1, IgG2, IgG3, and IgG4 isotype. In certainembodiments, the antibody is an IgG1 isotype.

In some embodiments of the methods disclosed herein, the antibody orfragment thereof is not covalently modified by the anti-reduction agent.

In some embodiments of the methods disclosed herein, the method does notincrease immunogenicity of the antibody or fragment thereof.

In some embodiments, the antibody is recombinantly expressed in the hostcell. In some embodiments, the antibody is selected from the groupconsisting of a therapeutic antibody, a modified antibody and aconjugated antibody.

In another aspect, the disclosure provides a cell culture, harvest orpre-harvest composition comprising an effective amount of ananti-reduction agent selected from the group consisting of methyleneblue, a quinone, a disulfide, a salt thereof and any combinationsthereof. In some embodiments, the quinone is at least one of asubstituted benzoquinone; 1,2-naphthoquinone-4-sulfonic acid; andanthraquinone-2-sulfonic acid; a coenzyme Q analog, and combinationsthereof. In some embodiments, the substituted benzoquinone isrepresented by formula (I):

wherein R₁, R₂, R₃, and R₄ is each independently selected from the groupconsisting of H, alkyl, alkoxy, COOH, and SO₃H.

In some embodiments, the coenzyme Q analog is one or more selected fromthe group consisting of coenzyme Q0, coenzyme Q2, and combinationsthereof.

In some embodiments, the disulfide is one or more selected from thegroup consisting of disulfiram; lipoic acid; a soluble cystine analog; acombination of glutathione reductase and oxidized glutathione (GSSG);oxidized glutathione alkyl esters; oxidized glutathione methyl esters;oxidized glutathione ethyl esters; oxidized glutathione isopropylesters; 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and any combinationsthereof.

In some embodiments, the cystine analog is one or more selected from thegroup consisting of cystine dimethyl ester, cystine diethyl ester,cystine bis(t-butyl) ester, cystine methyl ester, cystine ethyl ester,cystine t-butyl ester, di-N-acetyl cystine, L-cystine bis(methyl ester),L-cystine bis(t-butyl ester), and any combinations thereof.

In some embodiments, the composition comprises one or more selected fromthe group consisting of a mixture of anthraquinone-2-sulfonic acid andcystine dimethyl ester; a mixture of anthraquinone-2-sulfonic acid andcystine bis(t-butyl) ester; a mixture of lipoic acid andanthraquinone-2-sulfonic acid; a mixture of lipoic acid and cystinedimethyl ester; a mixture of lipoic acid and cystine bis(t-butyl) ester,and any combinations thereof.

In certain embodiments, the anti-reduction agent does not covalentlymodify the antibody or fragment thereof. In some embodiments, theanti-reduction agent is at a sub-stoichiometric concentration. In someembodiments, the anti-reduction agent is present at a concentration fromabout 0.01 mM to about 100 mM. In further embodiments, theanti-reduction agent is present at a concentration from about 0.1 mM toabout 10 mM.

In another aspect, the disclosure provides a cell culture, harvest orpre-harvest composition comprising an effective amount of one or moreselected from the group consisting of methylene blue; a substitutedbenzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; lipoic acid; disulfiram; a solublecystine analog; a combination of glutathione reductase and oxidizedglutathione (GSSG); oxidized glutathione alkyl esters; oxidizedglutathione alkyl esters; oxidized glutathione methyl esters; oxidizedglutathione ethyl esters; oxidized glutathione isopropyl esters; and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB).

In some embodiments of the methods and compositions disclosed herein,the composition is cell culture media, harvest cell culture fluid, orpre-harvest cell culture fluid.

Another aspect of this disclosure provides method of inhibitingdisulfide bond reduction in a recombinant protein, antibody, or fragmentthereof expressed in a host cell. The method comprises adding ananti-reduction agent to a cell culture media, pre-harvest cell culturefluid, or harvest cell culture fluid, comprising the antibody orfragment thereof, wherein the anti-reduction agent is an organic redoxactive anti-reduction agent or an inorganic redox active anti-reductionagent. In some embodiments, the anti-reduction agent is selected fromthe group consisting of ZnSO₄, CuSO₄, NiSO₄, NaNO₃, and any combinationsthereof.

A further aspect of this disclosure provides a method of inhibitingdisulfide bond reduction or fragmentation in an antibody, antibodyfragment, or recombinantly expressed protein. The method comprisescontacting the antibody, antibody fragment, or recombinantly expressedprotein with an anti-reduction agent, wherein the anti-reduction agentis an organic redox active anti-reduction agent or an inorganic redoxactive anti-reduction agent. In some embodiments, the anti-reductionagent is selected from the group consisting of ZnSO₄, CuSO₄, NiSO₄,NaNO₃, and any combinations thereof, wherein the antibody, antibodyfragment, or recombinantly expressed protein is contacted duringexpression in a host cell, during cell culture, pre-harvest, or harvest.

Yet another aspect of this disclosure provides a method of increasingproduction of a protein, antibody, or antibody fragment with intactnative disulfide bonds that is expressed in a mammalian host cell, themethod comprising adding an effective amount of an anti-reduction agentto a cell culture media, pre-harvest cell culture fluid, or harvest cellculture fluid, comprising the antibody or fragment thereof, wherein theanti-reduction agent is an organic redox active anti-reduction agent oran inorganic redox active anti-reduction agent. In some embodiments, theanti-reduction agent is selected from the group consisting of ZnSO₄,CuSO₄, NiSO₄, NaNO₃, and any combinations thereof.

In some embodiments of the methods disclosed herein, greater than about80% of the protein, antibody, or antibody fragment produced has intactnative disulfide bonds. In further embodiments, greater than about 90%of the protein, antibody, or antibody fragment produced has intactnative disulfide bonds.

In some embodiments of the methods disclosed herein, the methodcomprises adding the anti-reduction agent to the cell culture media. Insome embodiments, the method comprises adding the anti-reduction agentto the cell culture medium within about 48 hours of harvesting the cellculture. In some embodiments, the method comprises adding theanti-reduction agent to the cell culture medium within about 24 hours ofharvesting the cell culture. In some embodiments, the method comprisesadding the anti-reduction agent to the cell culture medium within about12 hours of harvesting the cell culture. In some embodiments, the methodcomprises adding the anti-reduction agent to the cell culture mediumwithin about 15 minutes of harvesting the cell culture. In someembodiments, the method comprises adding the anti-reduction agent to thepre-harvest cell culture fluid.

In some embodiments of the methods disclosed herein, the methodcomprises adding the anti-reduction agent to the harvest cell culturefluid.

In some embodiments of the methods disclosed herein, the anti-reductionagent is added to a final concentration in a range from about 0.01 mM toabout 100 mM.

In some embodiments of the methods disclosed herein, the mammalian hostcell is a Chinese Hamster Ovary (CHO) cell.

In some embodiments of the methods disclosed herein, the antibody isselected from an anti-FOLR1 antibody, an anti-CD56 antibody, ananti-CD37 antibody, an anti-EGFR antibody, an anti-IGF-1R antibody, ananti-MUC1, an anti-CA6 glycotope, an anti-CD19 antibody, and ananti-CD33 antibody. In some embodiments, the anti-FOLR1 antibodycomprises a heavy chain or light chain variable region sequencerepresented by SEQ ID NO.: 3, 4, or 5. In some embodiments, theanti-FOLR1 antibody is huMov19. In further embodiments, the anti-FOLR1antibody is huN901.

Another aspect of this disclosure provides a cell culture, harvest, orpre-harvest composition comprising an effective amount of ananti-reduction agent, wherein the anti-reduction agent is an organicredox active anti-reduction agent or an inorganic redox activeanti-reduction agent. In some embodiments, the anti-reduction agent isselected from the group consisting of ZnSO₄, CuSO₄, NiSO₄, NaNO₃, andany combinations thereof. In some embodiments the agent is an organicredox active anti-reduction agent. In some embodiments, the agent is aninorganic redox active anti-reduction agent. In some embodiments, theanti-reduction agent is present at a concentration ranging from about0.01 mM to about 100 mM. In some embodiments, the anti-reduction agentis present at a concentration ranging from about 0.1 mM to about 10 mM.In some embodiments, the anti-reduction agent is present at aconcentration of about 1 mM.

In some embodiments, the composition is cell culture media, harvest cellculture fluid, or pre-harvest cell culture fluid.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “antibody” or “antibodies” is meant an immunoglobulin molecule thatrecognizes and specifically binds to a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term “antibody” encompasses intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain antibodies, linear antibodies, diabodies (dAb), singledomain heavy chain antibodies, a single domain light chain antibodies,single chain Fv (scFv), multispecific antibodies such as bispecificantibodies generated from at least two intact antibodies, chimericantibodies, humanized antibodies, human antibodies, fusion proteinscomprising an antigen binding portion of an antibody, and any othermodified antibody or immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. In one example, the modified antibody is a probodyor an antibody or antibody fragment coupled to a masking moiety or acleavable moiety, wherein the masking moiety or cleavable moiety iscapable of being removed, cleaved, reduced or photolysed. In anotherexample the modified antibody is an antibody that includes a sitespecific (e.g., N-terminus, C-terminus, or cysteine modified orengineered) modification. An antibody can be of any the five majorclasses of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), basedon the identity of their heavy-chain constant domains referred to asalpha, delta, epsilon, gamma, and mu, respectively. The differentclasses of immunoglobulins have different and well known subunitstructures and three-dimensional configurations. Antibodies can be nakedor conjugated to other molecules such as toxins or radioisotopes. In oneexample, the antibody is conjugated to a cytotoxic agent (e.g., amaytansinoid) to form an antibody-drug-conjugate (ADC).

By “anti-folate receptor 1 (FOLR1) antibody” is meant an antibody orfragment thereof that specifically binds a folate receptor 1polypeptide. Non-limiting examples of an anti-FOLR1 antibody includemov19 and humanized (e.g., CDR grafted or resurfaced) versions thereof(huMov19″). The sequences for exemplary anti-FOLR1 antibodies aredisclosed, for example, in U.S. Pat. No. 8,557,966, and in U.S. PatentPublication Nos. 2012/0282175 and 2012/0009181, each of which isincorporated by reference herein in its entirety. In particularembodiments, the anti-FOLR1 antibody comprises a variable heavy chainand/or variable light chain that is substantially identical (e.g., atleast about 85%, 90%, 95%) to one of the following exemplary sequences:

huMov19 vHC SEQ ID NO: 3QVQLVQSGAEVVKPGASVKISCKASGYTFTGYFMNWVKQSPGQSLEWIGRIHPYDGDTFYNQKFQGKATLTVDKSSNTAHMELLSLTSEDFAVYYCTRYD GSRAMDYWGQGTTVTVSShuMov19 vLCv1.00 SEQ ID NO: 4DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLTYRASNLEAGVPDRFSGSGSKTDFTLNISPVEAEDAATYYCQQSREYPY TFGGGTKLEIKRhuMov19 vLCv1.60 SEQ ID NO: 5DIVLTQSPLSLAVSLGQPAIISCKASQSVSFAGTSLMHWYHQKPGQQPRLLTYRASNLEAGVPDRFSGSGSKTDFTLTISPVEAEDAATYYCQQSREYPY TFGGGTKLEIKR

By “anti-CD56 antibody” is meant an antibody or fragment thereof thatspecifically binds a CD56 polypeptide. One example of an anti-CD56antibody is the N901 antibody and humanized (e.g., CDR grafted andresurfaced) versions thereof. The preparation and exemplary sequences ofversions of humanized N901 (“huN901”), are described, for example, byRoguska et al, Proc. Natl. Acad. Sci. USA, 91:969-973 (1994), andRoguska et al, Protein Eng., 9:895:904 (1996), the disclosures of whichare incorporated by reference herein in their entirety.

To denote a humanized antibody, the letters “hu” or “h” appear beforethe name of the antibody. For example, humanized N901 may be referred toas huN901 or hN901. The sequences for huN901 are disclosed, for example,in U.S. Patent Publication No. 2012/0269827 which is incorporated byreference herein in its entirety. Exemplary N901 sequences are providedbelow.

N901LCv1.1 light chain 1 SEQ ID NO: 6DVVMTQSPLSLPVTLQPASISCRSSQIIIHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECN901HCv1.1 heavy chain 2 SEQ ID NO: 7QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSFTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMRKGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKOther exemplary sequences of human N901 are  provided below: gN901LCv1.1SEQ ID NO: 8 DVVMTQSPLSLPVTLGQPASISCRSSQIIIHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECgN901HCv1.1 SEQ ID NO: 9QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSFTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMRKGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbon atoms) and includes straight, branched chain, orcyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, such as, but notlimited to, ethyl, methyl, isopropyl, isobutyl, n-pentyl, n-hexyl andcyclopropylmethyl.

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃)alkoxy, such as, but not limited to,ethoxy and methoxy.

“Anti-reduction agent” refers to any small molecule compound that iscapable of minimizing reduction of disulfide groups in other molecules,such as antibodies or recombinant proteins. Anti-reduction agents of thepresent invention are useful to lower the thiol to antibody ratio,minimize disulfide bond reduction of an antibody or fragment thereof,retain intact native disulfide bonds of an antibody or fragment thereof,and/or increase a ratio of non-reduced to reduced antibody, or fragmentthereof. Some examples of anti-reduction agents include methylene blue;a quinone such as a substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; anthraquinone-2-sulfonic acid; and acoenzyme Q analog; and a disulfide such as a disulfiram; lipoic acid; asoluble cystine analog; a combination of glutathione reductase andoxidized glutathione (GSSG); and 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB). A substituted benzoquinone can include such structures asrepresented by formula I:

where R₁, R₂, R₃, and R₄ is each independently selected from H, alkyl,alkoxy, COOH, and SO₃H. Coenzyme Q analogs include such examples ascoenzyme Q0, coenzyme Q2, and combinations thereof. Cystine analogs caninclude cystine, cystine dimethyl ester, cystine diethyl ester, cystinemethyl ester, cystine ethyl ester, di-N-acetyl cystine, cystinebis(t-butyl ester) (CDBE), monoesters of cystine, asymmetric esters ofcystine, and any combinations thereof. In one embodiment, the cystineanalog is L-cystine bis (t-butyl ester).

By “cell culture” is meant the in vitro growth of cells.

By “cell culture medium” or “cell culture media” is meant a solutionused during culturing, growth, or maintenance of a cell. Exemplary cellsare mammalian host cells.

As used herein, the term “cystine” refers to a dimer of cysteine or aderivative thereof. Cystines may be asymmetric (i.e., mixed, wherein thetwo cysteines in the cystine are not identical) or symmetric (i.e.,wherein the two cysteines in the cystine are identical). As used herein,a cystine refers to a dimer of two L-cysteines or derivatives thereof, adimer of two D-cysteines or derivatives thereof, a dimer of oneL-cysteine or a derivative thereof and one D-cysteine or a derivativethereof, and any combinations thereof. In certain embodiments, thecystine is a dimer of two L-cysteines or derivatives thereof. In otherembodiments, the cystine is a dimer of two D-cysteines or derivativesthereof. In yet other embodiments, the cystine is a dimer of oneL-cysteine or a derivative thereof and one D-cysteine or a derivativethereof. In still other embodiments, where the cystine is L-cystinebis(t-butyl) ester (CDBE) or cystine dimethyl ester (CDME) the cystineis not sourced from animals and is transmissible spongiformencephalopathy (TSE) safe. In other embodiments, the cystine isanimal-derived cystine dimethyl ester (CDME), but is non-rodent derivedand is TSE safe. In still other embodiments, custom synthesis is carriedout using non-animal L-cystine.

The term “pre-harvest cell culture fluid” refers to the solution presentafter cell culture and before cell harvest. A pre-harvest cell culturefluid includes, but is not limited to, cell culture medium to which oneor more agents of the invention are optionally added. Pre-harvest marksthe beginning of cell harvesting operations when culture conditions areno longer optimized for cell growth. The cell culture media and/orpre-harvest cell culture fluid may contain proteins or antibodies thatare released (e.g., secreted) into the media or solution by the cellsduring culturing. Cell culture media is optimized for cell growth,whereas the pre-harvest and harvest cell culture fluids are optimizedfor cell separation and antibody purification. For example, thepre-harvest step can include preparation of the culture for harvest byreducing temperature, changing the pH (usually lowering to a pH of about5 to a pH of less than about 7), adding anti-reduction agents, such asvia the pumps that add feed media during culture, and flocculation. Thepre-harvest step can be optional as the cell culture media can be pumpeddirectly from the bioreactor where the cells are being cultured to thecentrifuge or filter for the harvesting step.

“Harvest cell culture fluid” refers to the solution present during thecell separation process and after separation of the cells from the cellculture media via methods, such as centrifugation or filtration. Aharvest cell culture fluid typically includes antibodies or recombinantproteins secreted by the cells during cell culture. A harvest cellculture fluid includes, but is not limited to cell culture medium towhich one or more anti-reduction agents of the invention are optionallyadded.

By “disulfide” is meant a compound with a linked pair of sulfur atoms.Examples of a disulfide include, but are not limited to, disulfiram,lipoic acid, a soluble cystine analog, a combination of glutathionereductase and oxidized glutathione (GSSG), oxidized glutathione alkylesters (including methyl esters, ethyl esters, and isopropyl esters),and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). A “disulfide bond”refers to one or more linked pairs of sulfur atoms or a covalent linkageof two thiol groups in a compound, antibody, or fragment thereof.

By “effective amount” is meant an amount of an anti-reduction agent ofthe invention sufficient to minimize disulfide bond reduction.Preferably disulfide bond reduction is minimized by at least about 10%,20%, 25%, 50%, 75%, or by 100%, such that it is virtually undetectableas compared to an untreated sample.

By “fragment” is meant a portion of an antibody, antibody molecule, orprotein molecule. This portion contains, preferably, at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of theantibody molecule. A fragment may contain 10, 20, 30, 40, 50, 60, 70,80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 aminoacids of the antibody.

“Fragmentation” refers to cleavage of the immunoglobulin molecule intofragments or smaller portions than the original expressed molecule. Thefragmentation phenomenon can occur during the antibody productionprocess, such as the use of excessive mechanical cell shear thatreleases reducing agents that reduce the antibody's interchain disulfidebonds during culture or harvest, or proteases that digest or cleavecertain portions of the immunoglobulin protein structure. Measurementsof fragmentation, such as thiol to antibody ratio, can be visualized ona gel, or by measuring thiol amount in the antibody by Ellman's assay orby an HPLC assay using derivatization of thiol.

By “large scale” or “production scale” is meant 80 L, 200 L, 500 L, 1200L, 600 L, and 12,000 L and various numbers in between.

By “minimizing a disulfide bond reduction” is meant decreasing the thiolto antibody ratio by more than about 25%, more than about 50%, more thanabout 75%, more than about 80%, more than about 85%, more than about90%, more than about 95%, more than about 98%, more than about 99%, ormore. Minimizing also refers to decreasing the percentage offragmentation by more than about 25%, 50%, 75%, 80%, 85%, 90%, 95%, 98%,99%, or more. Minimizing also refers to decreasing the amount ofnon-intact antibody, or retaining intact antibody, present at any stagein the purification process by at least about 50% of the total antibody(intact antibody+reduced antibody fragments), at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99%, or more. Measurements ofminimizing thiol to antibody ratio, decreasing fragmentation, ordecreasing non-intact antibody can be assessed from assays such as, forexample, quantification of antibody fragmentation on a gel.

By “quinone” is meant oxidized aromatic compounds. For example, someknown quinones include substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; and anthraquinone-2-sulfonic acid;coenzyme Q0 and coenzyme Q2-3.

By “reduction” is meant the cleavage of a disulfide bond in a protein,such as an antibody, or fragment thereof by a reducing agent.

By “sparging” is meant the addition of air or dissolved O₂ to the cellculture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid to achieve an O₂ concentration of at least about 20% toabout 100%. Sparging with O₂ can include achieving a dissolvedconcentration of greater than about 20%, or between about 20% to about100%. Sparging with O₂ also includes increasing the percentage of O₂saturation in the cell culture media, pre-harvest cell culture fluid,and/or harvest cell culture fluid to be in a range of about 100% of airsaturation (about 20% O₂) to about 500% of air saturation (about 100%O₂) (e.g., 100%, 110%, 120%, 125%, 130%, 140%, 150%, 160%, 170%, 175%,180%, 190%, 200%, 225%, 250% air saturation). In other embodiments, airsaturation ranges in the cell culture media, pre-harvest cell culturefluid, and/or harvest cell culture fluid are between about 100-125%,100-150%, 125-150%, 150-200%, and 200-250%.

“Sub-stoichiometric” refers to a molar concentration of the agent thatis less than the molar concentration of total thiol in a solution.

By “analog” is meant a molecule that is not identical, but has analogousfunctional or structural features. For example, a polypeptide analogretains the biological activity of a corresponding naturally-occurringpolypeptide, while having certain biochemical modifications that enhancethe analog's function relative to a naturally occurring polypeptide.Such biochemical modifications could increase the analog's proteaseresistance, membrane permeability, or half-life, without altering, forexample, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The terms “isolated,” “purified,” or “biologically pure” refers tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, an antibodyor fragment thereof is purified if it is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized. Purity and homogeneity are typically determined usinganalytical chemistry techniques, for example, polyacrylamide gelelectrophoresis or high performance liquid chromatography. The term“purified” can denote that the antibody or fragment thereof gives riseto essentially one band in an electrophoretic gel.

By an “isolated antibody or fragment thereof” is meant an antibody orfragment thereof of the invention that has been separated fromcomponents that naturally accompany it. Typically, the antibody orfragment thereof is isolated when it is at least 60%, by weight, freefrom the cellular proteins and naturally-occurring organic moleculeswith which it is naturally associated. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight, an antibody or fragment thereof of the invention. Anisolated antibody or fragment thereof of the invention may be obtained,for example, by extraction from a natural source, by recombinantexpression; or by chemical synthesis. Purity can be measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, or by HPLC analysis.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

The methods of producing the antibody or fragment thereof of theinvention in general comprise large scale or production scale of theantibody or fragment thereof, such that the antibody or fragment thereofis a therapeutic antibody or fragment thereof for administration to asubject (e.g., animal, human) in need thereof, including a mammal,particularly a human.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows treatment of humanized anti-FOLR1 IgG1antibody with damaged CHO cell supernatant with or without methyleneblue (MB) at 0.025 mM and 0.05 mM for 2.2 hours, followed by measurementof thiol per antibody.

FIG. 2 shows the mass spectrum of a deglycosylated, humanized antibodysample upon treatment with 0.05 mM methylene blue and damaged CHO cellsupernatant for 2.2 hours.

FIG. 3 is a graph that shows the effect of coenzyme Q analogs, whichprotect against the reduction of disulfide bonds in humanized N901 IgG1by damaged CHO cell lysate.

FIG. 4 is a graph that shows the effect of 0.025 mM, 0.05 mM, and 0.1 mM1,2-naphthoquinone-4-sulfonic acid (NQS), which protects against thereduction of antibody disulfide bonds by a CHO cell lysate.

FIG. 5 is a graph that shows the effect of 0.2 mManthraquinone-2-sulfonic acid (AQS), which protects against thereduction of disulfide bonds in an antibody by a CHO cell lysate;

FIG. 6A shows the mass spectrum of deglycosylated antibody sample fromantibody sample treated with anthraquinone-2-sulfonic acid (AQS) and aCHO cell lysate.

FIG. 6B shows the mass spectrum of deglycosylated antibody sample fromcontrol, unreduced humanized IgG1 antibody sample.

FIG. 7 is a graph that shows the effect of lipoic acid treatment, whichprotects native disulfide bonds in humanized IgG1 antibody exposed todamaged CHO cell supernatant.

FIG. 8A shows the mass spectrum of deglycosylated antibody sample fromantibody sample treated with lipoic acid and CHO cell lysate.

FIG. 8B shows the mass spectrum of deglycosylated antibody sample fromunreduced humanized IgG1 antibody sample.

FIG. 9 is a graph that shows the effect of L-cystine dimethyl ester(CDME), which protects against the reduction of disulfide bonds inantibody exposed to a CHO cell lysate;

FIG. 10 is a graph that shows the effect of L-cystine dimethyl ester(CDME) and L-cystine diethyl ester (CDEE), which protect against thereduction of disulfide bonds in an antibody by a CHO cell lysate.

FIG. 11 is a graph that shows the effect of a combination of L-cystinedimethyl ester

(CDME) and anthraquinone-2-sulfonic acid (AQS), which protects againstthe reduction of disulfide bonds in antibody by a CHO cell lysate.

FIGS. 12A and 12B show the effect of a combination of L-cystine dimethylester (CDME) and anthraquinone-2-sulfonic acid (AQS) on antibodyfragmentation in harvest cell culture fluid derived from humanizedIgG1-producing CHO cells. This combination is referred to as “AQC.”

FIG. 12A shows results of a non-reducing Protein Lab Chipelectrophoresis. FIG. 12B is a table showing the quantitative analysisof antibody fragmentation and intact antibody using a non-reducingProtein Lab Chip analysis. Samples treated at various time points with acombination of AQS and CDME (AQC) are compared to control sampleswithout any AQS or CDME added.

FIG. 13A shows the mass spectrum of deglycosylated antibody sample fromantibody sample treated with L-cystine dimethyl ester (CDME) and CHOcell lysate.

FIG. 13B shows the mass spectrum of deglycosylated antibody sample fromcontrol, unreduced humanized IgG1 antibody sample.

FIG. 14 is a graph that shows that L-cystine dimethyl ester (CDME) ismore effective than L-cystine for protection against the reduction ofdisulfide bonds in an antibody by a CHO cell lysate.

FIG. 15 is a graph that shows the effect of 2 mM L-cystine dimethylester (CDME), 2 mM anthraquinone-2-sulfonic acid (AQS), and theircombination (1 mM CDME+1 mM AQS), which protect against the reduction ofdisulfide bonds in an antibody by a CHO cell lysate.

FIG. 16 is a graph that shows the effect of 1 mM L-cystine dimethylester (CDME), 1 mM anthraquinone-2-sulfonic acid (AQS), and theircombination (1 mM CDME+1 mM AQS), which protect against thefragmentation of an antibody by a microfluidized CHO lysate (20% v/v).

FIG. 17 is a graph that shows the effect of 0.5 and 1 mM L-cystinebis(t-butyl) ester (CDBE), which protect against the reduction ofdisulfide bonds in an antibody by a CHO cell lysate.

FIG. 18 is a graph that shows the effect of a combination of glutathionereductase and oxidized glutathione (GSSG), which protects againstreduction of disulfide bonds in antibody by CHO cell lysate.

FIG. 19 is a graph that shows the effect of disulfiram, which protectsagainst the reduction of disulfide bonds in antibody by damaged CHO cellsupernatant.

FIG. 20 is a graph that shows the disulfide-protective effect of variouscombinations of L-cystine dimethyl ester (CDME) with air, oxygen, andnitrogen.

FIG. 21 is a graph that shows the disulfide-protective effect of 1 mMCDME or 1 mM CDBE treatment on a humanized IgG4 antibody.

FIG. 22 is a graph that shows the disulfide-protective effect of 1 mMCDME or 1 mM CDBE treatment on a humanized IgG2 antibody.

FIG. 23 is a graph that shows the effect of various additives on redoxpotential over a range of concentration of DTT.

FIG. 24 is a graph that shows the effect of various additives on percentof intact antibody over a range of concentration of DTT.

FIG. 25 is a graph that shows the percent intact antibody obtained whenThioredoxin Reductase is inhibited.

FIG. 26 is a graph showing the results of a biological assay usingvarious additives to protect M9346A from disulfide reduction.

FIG. 27 is a series of graphs showing the data that resulted from abiological assay with varying stringency parameters.

FIG. 28 is a graph showing the results of a stringency evaluation ofvarious media additives.

DETAILED DESCRIPTION OF THE INVENTION

As described below, the present invention features compositions andmethods for protecting against fragmentation and disulfide bondreduction in antibodies and other recombinant proteins.

The invention is based, at least in part, on the discovery of agents(e.g., methylene blue; a substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; anthraquinone-2-sulfonic acid;lipoic acid; disulfiram; a soluble cystine analog; a combination ofglutathione reductase and oxidized glutathione (GSSG); oxidizedglutathione alkyl esters (including methyl esters, ethyl esters, andisopropyl esters), and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)) thatminimize native disulfide bond reduction, thereby increasing theexpression and/or production of recombinant proteins, antibodies, orfragments thereof, with intact native disulfide bonds. Such agents areadvantageously non-toxic and do not result in the unintended covalentmodification of antibodies or recombinant proteins.

Recombinant Protein and/or Antibody Production

The production and purification of antibodies and recombinant proteinstypically includes cell separation step that can result in the releaseof intracellular reducing proteins and peptides containing thiol groups.Such reducing proteins and peptides likely contribute to the undesirablereduction of inter-chain disulfide bonds in antibodies and recombinantproteins. As reported herein below, a number of agents of the invention(e.g., methylene blue; a substituted benzoquinone;1,2-naphthoquinone-4-sulfonic acid; anthraquinone-2-sulfonic acid;lipoic acid; disulfiram; a soluble cystine analog; a combination ofglutathione reductase and oxidized glutathione (GSSG); oxidizedglutathione alkyl esters (including methyl esters, ethyl esters, andisopropyl esters), and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)) havebeen identified that minimize the reduction of antibodies andrecombinant proteins. These agents can be added to cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid atvirtually any point during the expression, production, and/orpurification of antibodies or other recombinant proteins, or fragmentsthereof, in a mammalian host cell.

Such anti-reduction agents (e.g., methylene blue; a substitutedbenzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; lipoic acid; disulfiram; a solublecystine analog; a combination of glutathione reductase and oxidizedglutathione (GSSG); oxidized glutathione alkyl esters (including methylesters, ethyl esters, and isopropyl esters), and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)) advantageously minimize orprevent native disulfide bonds reduction, thereby increasing theexpression and/or production of antibodies, or fragment thereof, havingintact native disulfide bonds. Such anti-reduction agents areparticularly advantageous because they do not result in the unintendedcovalent modification of the antibody, or fragment thereof, and wouldnot increase the immunogenicity of an antibody, or fragment thereof,used for therapeutic purposes. In addition, agents of the inventionunexpectedly decrease the extent of disulfide reduction even atconcentrations that are below the level of the total thiol in the cellculture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid.

Accordingly, the present invention provides cell culture compositionscomprising agents described herein (e.g., methylene blue; a substitutedbenzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; lipoic acid; disulfiram; a solublecystine analog; a combination of glutathione reductase and oxidizedglutathione (GSSG); oxidized glutathione alkyl esters (including methylesters, ethyl esters, and isopropyl esters), and5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and combinations thereof)that prevent or minimize protein or antibody reduction. The inventionfurther provides methods of using such cell culture compositions forculturing mammalian host cells that express recombinant proteins,antibodies, or fragments thereof.

Compositions

Compositions of the present invention are useful for minimizingreduction of a disulfide bond in a recombinant protein, antibody orfragment thereof that is expressed in a mammalian host cell. Thecompositions of the invention include cell culture media, pre-harvestcell culture fluid, and/or harvest cell culture fluid. Such compositionscan include aqueous compositions useful in the production of recombinantproteins, such as antibodies. Exemplary cell culture medias are listedin Table 4.

TABLE 4 Cell culture medias. Description Basal/Feed Media ManufacturerCD CHO Basal Media Life Technologies CD FortiCHO Basal Media LifeTechnologies CD Efficient Feed A Feed Media Life Technologies CDEfficient Feed B Feed Media Life Technologies CD Efficient Feed C FeedMedia Life Technologies ExCell 325 PF Basal Media SAFC ExCell CHOZNBasal Media SAFC Media ExCell CHOZN Feed Feed Media SAFC Hyclone CDM4CHOBasal Media ThermoFisher Hyclone Hycell Basal Media ThermoFisher HycloneCellBoost Feed Media ThermoFisher

The compositions include an agent of the invention, such as ananti-reduction agent, in an amount sufficient to minimize reduction of adisulfide bond. As described herein, the anti-reduction agent includesone or more of the following: methylene blue, a quinone, a disulfide, asalt thereof, and any combinations thereof.

Methylene Blue

Methylene blue is a heterocyclic aromatic chemical compound with amolecular formula of C₁₆H₁₈N₃SCl. See Table 1.

TABLE 1 Structural formula of methylene blue. Compound StructuralFormula Methylene blue

Recent studies have found that methylene blue may have a neuroprotectiveeffect. In low doses, methylene blue protects the brain from disease byacting as an antioxidant in the mitochondria. It functions as analternative mitochondrial electron transfer carrier to enhance cellularoxygen consumption and thus provide neuroprotection in vitro.

Compositions and methods for culturing mammalian host cells that expressan antibody or fragment thereof can include methylene blue as ananti-reduction agent in an amount sufficient to minimize reduction of adisulfide bond in the antibody or fragment thereof. In one embodiment,methylene blue can be added to a final concentration in the range ofabout 0.01 mM to about 100 mM (e.g., 0.01, 0.1, 0.5, 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), about0.05 mM to about 50 mM, and about 0.1 mM to about 10 mM. In particularembodiments, a composition of the invention (e.g., cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid)comprises about 0.025 and/or about 0.05 mM of methylene blue.

In yet another embodiment, methylene blue can be added at a molar ratioof about 0.01 to about 10 of the total thiol concentration in the cellculture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid, about 0.05 to about 2.5 of the total thiol concentration,about 0.07 to about 1 of the total thiol concentration, and/or about 0.1to about 0.8 of the total thiol concentration. In another embodiment,methylene blue can be added at sub-stoichiometric concentrations or to amolar concentration of the anti-reduction agent that is less than themolar concentration of total thiol in the cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid.

Quinones

Quinones are oxidized, conjugated compounds that are derived fromaromatic compounds, such as benzene or naphthalene. Quinones areelectrophilic acceptors that are stabilized by conjugation. They readilyreact with electron-donating substituents. Depending on the quinone andthe site of reduction, reduction can either re-aromatise the quinone orbreak the conjugation.

Some examples of specific quinones useful as anti-reduction agents inthe present invention include, but are not limited to, substitutedbenzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; coenzyme Q0, coenzyme Q2-3, a saltthereof, and any combinations thereof. In one particular embodiment, ananti-reduction agent of the invention is a Coenzyme Q analog, such as Q0(2,3-dimethoxy-5-methyl-p-benzoquinone) or Q2(2,3-dimethoxy-5-methyl-6-geranyl-p-benzoquinone).

A substituted benzoquinone can include such structures as represented byformula (I):

where R₁, R₂, R₃, and R₄ can be each independently selected from thegroup consisting of H, alkyl, alkoxy, COOH, and SO₃H. Coenzyme Q analogscan include such examples as coenzyme Q0, coenzyme Q2, and combinationsthereof. See Table 2 for structural formulas.

In one embodiment, the quinone is anthraquinone-2-sulfonic acid and iseffective at lowering the antibody disulfide reduction.

Quinone structural formulas. Compound Structural Formula Substitutedbenzoquinone

R_(1,2,3,4) = H, alkyl, alkoxy, COOH, SO₃H Coenzyme Q0

Coenzyme Q2

n = 2 1,2-Naphthoquinone-4- sulfonic acid

Anthraquinone-2- sulfonic acid

Compositions and methods for culturing mammalian host cells that expressan antibody or fragment thereof can include one or more quinones as ananti-reduction agent in an amount sufficient to minimize reduction of adisulfide bond in the antibody or fragment thereof. In one embodiment, aquinone can be added to a final concentration in the range of about 0.01mM to about 100 mM (e.g., 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), about 0.05 mM toabout 50 mM, and about 0.1 mM to about 10 mM. In particular embodiments,Q0 or Q2 (2,3-dimethoxy-5-methyl-6-geranyl-p-benzoquinone) is added to afinal concentration of about 0.2 or 0.1 mM.

In yet another embodiment, a quinone can be added at a molar ratio ofabout 0.01 to about 10 of the total thiol concentration in the cellculture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid, about 0.05 to about 2.5 of the total thiol concentration,about 0.07 to about 1 of the total thiol concentration, and about 0.1 toabout 0.8 of the total thiol concentration.

Disulfides

Disulfides are compounds containing a linked pair of sulfur atoms or adisulfide bond. Examples of disulfides include disulfiram; lipoic acid;a soluble cystine analog; a combination of glutathione reductase andoxidized glutathione (GSSG); oxidized glutathione alkyl esters(including methyl esters, ethyl esters, and isopropyl esters);5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), a salt thereof, and anycombinations thereof. Cystine analogs are also disulfides and includecystine dimethyl ester, cystine diethyl ester, cystine bis(t-butyl)ester, cystine methyl ester, cystine ethyl ester, cystine t-butyl ester,di-N-acetyl cystine, L-cystine bis(t-butyl ester), monesters of cystine,asymmetric esters of cystine, and any combinations thereof. Thedisulfides useful within the invention may also include symmetric andasymmetric disulfides of the sulfides recited herein. See Table 3 fordisulfide structural formulas.

TABLE 3 Disulfide structural formulas. Compound Structural FormulaCystine dimethyl ester

Cystine diethyl ester

Cystine bis(t-butyl) ester

Cystine methyl ester

Cystine ethyl ester

Cystine t-butyl ester

L-cystine bis(t-butyl ester)

Di-N-acetylcystine

Lipoic acid

Oxidized glutathione alkyl esters

Disulfiram (Tetraethylthiuram disulfide)

5,5′-Dithiobis(2- nitrobenzoic acid)

Disulfiram is commonly used as a treatment for chronic alcoholism byblocking the processing of alcohol in the body by inhibitingacetaldehyde dehydrogenase. Lipoic acid is a strained 5-member cyclicdisulfide. Cystine dimethyl ester (CDME) is a soluble, non-toxicdisulfide that is typically used to prevent kidney stones. Thedisulfides possess high potential to be reduced and can thereby protectdisulfide bonds in recombinant proteins, antibodies or fragmentsthereof. Lipoic acid contains a strained 5-membered cyclic disulfide (S.Sunner, Nature, 176, 217, 1955). The strained cyclic disulfide group inlipoic acid is unexpectedly more reactive toward thiol than non-cyclicdisulfides, which would favor the reduction of lipoic acid by CHO cellthiol proteins in comparison to antibody disulfide bonds. Oxidizedglutathione alkyl esters (including methyl esters, ethyl esters, andisopropyl esters), both symmetric and asymmetric, are also useful toprotect disulfide bonds in recombinant proteins, antibodies or fragmentsthereof.

Disulfides are also useful anti-reduction agents for their unexpectedsolubilities in water, in particular cystine dimethyl esterdihydrochloride, cystine diethyl ester dihydrochloride, and cystinebis(t-butyl) ester. In one embodiment, cystines useful within theinvention comprise L-cystine dimethyl ester dihydrochloride, L-cystinediethyl ester dihydrochloride, L-cystine bis(t-butyl) ester, cystinedimethyl ester, or any combinations thereof. Compositions and methodsfor culturing mammalian host cells that express an antibody or fragmentthereof can include one or more disulfides as an anti-reduction agent inan amount sufficient to minimize reduction of a disulfide bond in theantibody or fragment thereof. The disulfide can be added to a finalconcentration in the range of about 0.01 mM to about 100 mM (e.g., 0.01,0.1, 0.5, 1, 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100), about 0.05 mM to about 50 mM, and about0.1 mM to about 10 mM.

Inorganic and Organic Redox Active Substances

Certain inorganic and organic redox active substances are also usefulanti-reduction agents. These redox active substances can be used tomodulate the redox potential of cell culture media, pre-harvest cellculture fluid, or harvest cell culture fluid to protect antibodies fromdisulfide reduction. These substances have an aptitude to acceptelectrons greater than the redox potential of inter- and intrachaindisulfide bonds. As a result, they can neutralize reduction equivalents.Such substances include, but are not limited to, ZnSO₄, CuSO₄, NiSO₄,NaNO₃, and any combinations thereof. These substances can be added to afinal concentration in the range of about 0.01 mM to about 100 mM (e.g.,0.01, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100), about 0.05 mM to about 50 mM, andabout 0.1 mM to about 10 mM.

In one embodiment, the final concentration of one or more anti-reductionagents can be at least about 0.001 mM, 0.005 mM, 0.01 mM, 0.015 mM, 0.02mM, 0.025 mM, 0.03 mM, 0.035 mM, 0.04 mM, 0.045 mM, 0.05 mM, 0.055 mM,0.06 mM, 0.065 mM, 0.07 mM, 0.075 mM, 0.08 mM, 0.085 mM, 0.09 mM, 0.095mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM, 0.35 mM, 0.4 mM, 0.45 mM,0.5 mM, 0.7 mM, 1.0 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM,10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM,and any concentration in between. In another embodiment, one or moreanti-reduction agents can be added to a final concentration in the rangeof about 0.01 mM to about 100 mM, about 0.05 mM to about 50 mM, andabout 0.1 mM to about 10 mM. In a particular embodiment, one or more ofthe anti-reduction agents is added at a sub-stoichiometricconcentration. In another embodiment, the anti-reduction agent is at aconcentration of less than about 10 mM.

In yet another embodiment, one or more anti-reduction agents can beadded at a molar ratio of about 0.01 to about 10 of the total thiolconcentration in the cell culture media, pre-harvest cell culture fluid,and/or harvest cell culture fluid, about 0.05 to about 2.5 of the totalthiol concentration, about 0.07 to about 1 of the total thiolconcentration, and about 0.1 to about 0.8 of the total thiolconcentration.

In another embodiment, one or more anti-reduction agents can be added ata molar ratio of about 0.001, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03,0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085,0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.7, 0.8,0.9, and 1.0 of the total thiol concentration in the cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid.

The anti-reduction agents are also useful to lower a thiol:antibodyratio. The thiol:antibody ratio for the antibody or fragment thereof canbe lowered in the presence of anti-reduction agent by at least about10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% or more thanin the absence of the agent. In one particular embodiment, the antibodyor fragment thereof has a thiol:antibody ratio of at least about 25%lower in the presence of the anti-reduction agent than in the absence ofthe anti-reduction agent. In another particular embodiment, the antibodyor fragment thereof has a thiol:antibody ratio of at least about 50%lower in the presence of the anti-reduction agent than in the absence ofthe anti-reduction agent.

The ratio of non-reduced to reduced antibody or fragment thereof that isproduced by a mammalian host cell is increased by adding a sufficientamount of an anti-reduction agent to a cell culture media, pre-harvestcell culture fluid, and/or harvest cell culture fluid. The ratio can beincreased by at least about 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 12 fold, 15 fold, 17fold, or more. In one embodiment, the ratio is increased by at leastabout 2-fold. In another embodiment, the ratio is increased by at leastabout 10-fold.

As described above, the anti-reduction agent can include a combinationof methylene blue, one or more quinones, and/or one or more disulfides.In a particular embodiment, the composition includes a mixture ofanthraquinone-2-sulfonic acid and cystine dimethyl ester; a mixture oflipoic acid and anthraquinone-2-sulfonic acid; or a mixture of lipoicacid and cystine dimethyl ester.

It is also useful that the anti-reduction agent does not covalentlymodify the antibody or fragment thereof. Covalent modification couldincrease the immunogenicity of antibody and could also adversely affectthe physicochemical behavior of the antibody. Thus, after treatment withthe anti-reduction agent of the invention, the antibody or fragmentthereof substantially possesses its native folded structure and retainsits antigen binding site without alteration.

Also useful are anti-reduction agents that do not detectably orsubstantially decrease viability of the host cells. In one embodiment,the anti-reduction agent of the present invention does not decreaseviability by greater than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, or less. In one embodiment, the anti-reduction agentdoes not decrease viability of the cells by greater than about 15%.

The compounds useful within the invention may possess one or morestereocenters, and each stereocenter may exist independently in eitherthe (R) or (S) configuration. In one embodiment, compounds describedherein are present in optically active or racemic forms. The compoundsdescribed herein encompass racemic, optically active, regioisomeric andstereoisomeric forms, or combinations thereof that possess the usefulproperties described herein. Preparation of optically active forms isachieved in any suitable manner, including by way of non-limitingexample, by resolution of the racemic form with recrystallizationtechniques, synthesis from optically-active starting materials, chiralsynthesis, or chromatographic separation using a chiral stationaryphase. In one embodiment, a mixture of one or more isomer is utilized inthe composition described herein. In another embodiment, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

The methods described herein include the use of crystalline forms (alsoknown as polymorphs), solvates, amorphous phases, and/or acceptablesalts of compounds having the structure of any compound useful withinthe invention, as well as derivatives thereof having the same type ofactivity. Solvates include water, ether (e.g., tetrahydrofuran, methyltert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and thelike. In one embodiment, the compounds described herein exist insolvated forms with acceptable solvents such as water, and ethanol. Inanother embodiment, the compounds described herein exist in unsolvatedform.

In one embodiment, the compounds of the invention may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

The compounds described herein may form salts with acids or bases, andsuch salts are included in the present invention. The term “salts”embraces addition salts of free acids and addition salts of free basesthat are useful within the methods of the invention.

Suitable acceptable acid addition salts may be prepared from aninorganic acid or from an organic acid. Examples of inorganic acidsinclude hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric(including sulfate and hydrogen sulfate), and phosphoric acids(including hydrogen phosphate and dihydrogen phosphate). Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, examples of which include formic, acetic, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic,glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic,mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic,benzenesulfonic, pantothenic, trifluoromethanesulfonic,2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic,cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic,galactaric and galacturonic acid.

Suitable acceptable base addition salts of compounds of the inventioninclude, for example, metallic salts including alkali metal, alkalineearth metal and transition metal salts such as, for example, calcium,magnesium, potassium, sodium and zinc salts. Acceptable base additionsalts also include organic salts made from basic amines such as, forexample, N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. All of these salts may be prepared from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

Antibody Production

Described herein are methods for minimizing disulfide bond reduction orfragmentation of an antibody or fragment thereof that is expressed in amammalian host cell. As disulfide bond reduction and fragmentation canoccur during multiple stages of the antibody production process, themethods described herein address these issues by adding a sufficientamount of an anti-reduction agent to a cell culture media, pre-harvestcell culture fluid, and/or harvest cell culture fluid.

In one aspect, a method is provided for minimizing disulfide bondreduction in an antibody or fragment thereof that is expressed in amammalian host cell includes adding a composition comprising one or moreagents of the invention (e.g., methylene blue; a substitutedbenzoquinone; 1,2-naphthoquinone-4-sulfonic acid;anthraquinone-2-sulfonic acid; lipoic acid; disulfiram; a solublecystine analog; a combination of glutathione reductase and oxidizedglutathione (GSSG); and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)) to acell culture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid. The anti-reduction agent is one or more of methyleneblue, a quinone, a disulfide, a salt thereof and any combinationsthereof.

In another aspect, a method is provided for increasing production of anantibody or fragment thereof with intact native disulfide bonds that isexpressed in a mammalian host cell. The method includes adding asufficient amount of an anti-reduction agent to a cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid, wherethe anti-reduction agent is at least one of methylene blue, a quinone, adisulfide, a salt thereof, and any combinations thereof.

In yet another aspect, a method of increasing a ratio of non-reduced toreduced antibody or fragment thereof that is produced by a mammalianhost cell is described. The method includes adding a sufficient amountof an anti-reduction agent, such as one or more of methylene blue, aquinone, a disulfide, a salt thereof, and any combinations thereof, to acell culture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid. In one embodiment, the method increases the ratio by atleast about 2-fold. In another embodiment, the ratio is increased by atleast about 10-fold.

The methods provide for adding the anti-reduction agent at variousstages in the production process. In one embodiment, the anti-reductionagent is added to the cell culture medium. The anti-reduction agent canbe added within about 15 minutes of harvesting the cell culture, about30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36hours, 48 hours, or any time point in between. In a particularembodiment, the anti-reduction agent is added to the cell culture mediumwithin 48 hours of harvesting the cell culture. In another particularembodiment, the anti-reduction agent is added to the cell culture mediumwithin 24 hours of harvesting the cell culture. In yet anotherparticular embodiment, the anti-reduction agent is added to the cellculture medium within 12 hours of harvesting the cell culture.

In another embodiment, the anti-reduction agent is added to thepre-harvest cell culture fluid. In yet another embodiment, theanti-reduction agent is added to the harvest cell culture fluid.

The methods also provide for anti-reduction agents that do notsubstantially decrease viability of the host cells. Adding theanti-reduction agent does not decrease viability by greater than about30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less. In oneembodiment, adding the anti-reduction agent does not decrease viabilityof the cells by greater than about 15%.

Any mammalian host cell can be used with the methods and compositionsdescribed herein. Some examples include Chinese hamster ovary (CHO)cells, SP2/0, and NS0 cells.

In one embodiment, the antibody or fragment thereof includes an IgG1,IgG2, IgG3, and IgG4 isotype antibody. In another embodiment, theantibody or fragment thereof is an anti-FOLR1 antibody, an anti-CD56antibody, an anti-CD37 antibody, an anti-EGFR antibody, an anti-IGF-1receptor antibody, anti-muc1 (e.g., DS6-humanized or mouse), which isdescribed in WO2005/009369 and WO2007/024222, each of which isincorporated herein by reference in its entirety, an anti-CA6 glycotopeantibody, an anti-CD19 (e.g., B4 antibody (huB4 antibody), or ananti-CD33 antibody. Such antibodies are described in InternationalApplication Nos.: WO2004/043344, WO2003/106621, WO2005/061541,WO2011/112978, WO2012/058592, and WO2012/058588A2, which areincorporated by reference herein.

In one embodiment, an anti-FOLR1 antibody is an antibody that is capableof binding FOLR1 with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting FOLR1. Theextent of binding of an anti-FOLR1 antibody to an unrelated, non-FOLR1protein is less than about 10% of the binding of the antibody to FOLR1as measured, e.g., by a radioimmunoassay (MA) or ELISA. Anti-FOLR1antibodies are known in the art and are disclosed, for example, in U.S.Pat. No. 8,557,966 and US Appl. Pub. Nos. 2012/0282175 and 2012/0009181,each of which is herein incorporated by reference in its entirety.

The full-length amino acid (aa) and nucleotide (nt) sequences for FOLR1are known in the art and are provided below:

human folate receptor 1 amino acid sequence SEQ ID NO: 1MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLAL MLLWLLShuman folate receptor 1 nucleic acid sequence SEQ ID NO: 2atggctcagcggatgacaacacagctgctgctccttctagtgtgggtggctgtagtaggggaggctcagacaaggattgcatgggccaggactgagcttctcaatgtctgcatgaacgccaagcaccacaaggaaaagccaggccccgaggacaagttgcatgagcagtgtcgaccctggaggaagaatgcctgctgttctaccaacaccagccaggaagcccataaggatgtttcctacctatatagattcaactggaaccactgtggagagatggcacctgcctgcaaacggcatttcatccaggacacctgcctctacgagtgctcccccaacttggggccctggatccagcaggtggatcagagctggcgcaaagagcgggtactgaacgtgcccctgtgcaaagaggactgtgagcaatggtgggaagattgtcgcacctcctacacctgcaagagcaactggcacaagggctggaactggacttcagggtttaacaagtgcgcagtgggagctgcctgccaacctttccatttctacttccccacacccactgttctgtgcaatgaaatctggactcactcctacaaggtcagcaactacagccgagggagtggccgctgcatccagatgtggttcgacccagcccagggcaaccccaatgaggaggtggcgaggttctatgctgcagccatgagtggggctgggccctgggcagcctggcctttcctgcttagcctggcccta atgctgctgtggctgctcagc

A specifically useful antibody for detection of FOLR1 is the mousemonoclonal anti-huFOLR1 clone BN3.2 (Leica # NCL-L-FRalpha). An exampleof a therapeutically effective anti-FOLR1 antibody is huMov19 (M9346A).The polypeptides of SEQ ID NOs: 3-5 comprise the variable domain of theheavy chain of huMov19 (M9346A), and the variable domain light chainversion 1.00, the variable domain light chain version 1.60 of huMov19,respectively. In certain embodiments, the huMov19 (M9346A) antibody isencoded by the plasmids deposited with the American Type CultureCollection (ATCC), located at 10801 University Boulevard, Manassas, Va.20110 on Apr. 7, 2010 under the terms of the Budapest Treaty and havingATCC deposit nos. PTA-10772 and PTA-10773 or 10774. Also included areany of the anti-FOLR1 antibodies described in U.S. ProvisionalApplication No. 61/875,475, filed on Sep. 9, 2013. In an exemplaryembodiment, the 353-2.1 antibody is included as described therein.

In another particular embodiment, the anti-CD56 antibody is huN901. TheCD56 antigen is a neural cell adhesion molecule (NCAM) that is expressedon the surface of tumor cells of neuroendocrine origin, including smallcell lung carcinomas (SCLC), carcinoid tumors and Merkel cell carcinomas(MCC). CD56 is expressed on approximately 56% of ovarian tumors. See,e.g., Whiteman, K. R. et. al., AACR Annual Meeting, Abstract No. 2135,“Preclinical Evaluation of IMGN901 (huN901-DM1) as a PotentialTherapeutic for Ovarian Cancer” (April 2008). CD56 is also expressed onapproximately 70% of multiple myelomas. See, e.g., Tassone, P. et al.,Cancer Res. 64:4629-4636 (2004).

The preparation of different versions of humanized N901, is described,for example, by Roguska et al, Proc. Natl. Acad. Sci. USA, 91:969-973(1994), and Roguska et al, Protein Eng., 9:895:904 (1996), thedisclosures of which are incorporated by reference herein in theirentirety. In particular embodiments, an humanized N901 antibodycomprises or consists of a sequence described in Roguska supra. Todenote a humanized antibody, the letters “hu” or “h” appear before thename of the antibody. For example, humanized N901 may be referred to ashuN901 or hN901. The sequences for huN901 are disclosed, for example, inU.S. Patent Publication No. 2012/0269827 which is incorporated byreference herein in its entirety.

Sparging

Sparging is a technique of infusing, such as bubbling, gas through aliquid. A gas can be introduced into the liquid in the form of smallbubbles. The sparging device is typically fabricated with smallapertures through which gas is injected into the liquid, to provide arelatively fine dispersion of gas bubbles in the liquid undergoingtreatment. In some systems, the sparger can be positioned at the bottomof the culture so that the small gas bubbles rise slowly through theliquid to provide an extended period of gas-liquid contact.

The present invention includes sparging the cell culture media,pre-harvest cell culture fluid, and/or harvest cell culture fluid withoxygen, O₂. In one aspect, a method of minimizing disulfide bondreduction in an antibody or fragment thereof that is expressed in amammalian host cell is provided. The method includes culturing the hostcell in a concentration of at least about 20% dissolved O₂.

Unlike methods that sparge with air, which can achieve only minimal O₂concentrations dissolved in the culture media, it has been discoveredthat high O₂ concentrations are important for preserving disulfide bondsand/or minimizing reduction of antibodies during the production process.Thus, sparging with O₂ or a combination of air supplemented with O₂ canachieve optimal concentrations of dissolved O₂, higher than thoseobtained with air sparging, to reduce and/or prevent antibodyfragmentation. In one embodiment, the dissolved O₂ concentration in thecell culture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid is in the range of at least about 20% to about 100% O₂.

The percentage of O₂ saturation in the cell culture media, pre-harvestcell culture fluid, and/or harvest cell culture fluid can be in a rangeof about 100% of air saturation (about 20% O₂) to about 500% of airsaturation (about 100% O₂) via sparging with O₂ gas. The percentage ofO₂ saturation can be in a range of about 100 to about 125%, about 100 toabout 150%, about 125 to about 150%, about 150 to about 200%, about 200to about 250%, about 250 to about 300%, about 300 to about 350%, about350 to about 400%, about 400 to about 450%, and about 450% to about 500%of air saturation. The percentage of O₂ saturation in the cell culturemedia, pre-harvest cell culture fluid, and/or harvest cell culture fluidcan be about 100%, 110%, 120%, 125%, 130%, 140%, 150%, 160%, 170%, 175%,180%, 190%, 200%, 225%, 250%, 375%, 400%, 425%, 450%, 475%, and about500% of air saturation.

The use of sparging with O₂ can also be combined with the addition ofanti-reduction agents. In one embodiment, methods are included forminimizing disulfide bond reduction in an antibody or fragment thereofby adding a sufficient amount of one or more anti-reduction agents to acell culture media, pre-harvest cell culture fluid, and/or harvest cellculture fluid and culturing the host cell in a concentration of at leastabout 20% dissolved O₂.

Sparging with O₂ can also be employed on a large scale or productionscale to reduce fragmentation and/or minimize reduction of antibodies orfragments thereof during the large scale production process.

Therapeutic Antibodies or Fragments Thereof

In one aspect, a method is described for producing a therapeuticantibody, or fragment thereof, by exposing a mammalian host cell thatproduces the therapeutic antibody, or fragment thereof, to a compositionthat includes an anti-reduction agent of the invention in a cell culturemedia, pre-harvest cell culture fluid, and/or harvest cell culturefluid, wherein the anti-reduction agent is at least one of methyleneblue, a quinone, and a disulfide.

In another aspect, a method is provided for producing a therapeuticantibody, or fragment thereof, by exposing a mammalian host cell thatproduces the therapeutic antibody, or fragment thereof, to aconcentration of at least about 20% O₂. The O₂ concentration can beachieved via O₂ sparging as described herein.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991); and “Antibodies: ALaboratory Manual” (Harlow and Lane, 1988). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES

Antibodies and other proteins are often expressed recombinantly. In oneembodiment, a vector encoding an antibody or protein of interest istransferred to a host cell by conventional techniques and thetransfected cells are then cultured to produce the antibody orrecombinant protein. During the course of antibody production, celldamage can occur, particularly during the cell-separation process. Thisdamage can be simulated by methods that disrupt cells. For example, byexposing cells to multiple freeze-thaw cycles, by chemical means, forexample, by exposing the cells to detergents, or by mechanical means,for example, using a microfluidizer.

For all of the examples described below, cell damage was simulated bymultiple freeze-thaw cycles of cells, by addition of RIPA buffer (50 mMTris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodiumdodecylsulfate), or by using microfluidizer. This damaged cellsupernatant or lysate was then incubated with a buffered solutioncontaining antibody with or without the anti-reduction agent beingtested, following which the extent of the antibody-disulfide reductionwas measured using Ellman's assay for thiol (P. W. Riddles, R. L.Blakeley, and B. Zerner, Methods Enzymol., 91, 49-60, 1983) or bynon-reducing SDS protein LabChip analysis. The total thiol level presentin the mixture of damaged cell supernatant/lysate was measuredinitially. Unexpectedly, several substances were found to lower theantibody-disulfide reduction even upon addition at levels below thelevel of the total thiol present in the mixture of the damaged cellsupernatant/lysate and antibody.

Example 1: Methylene Blue Decreased Antibody Disulfide Bond Reduction

A simulated, damaged CHO cell suspension was prepared using 300 millionCHO cells per ml phosphate buffered saline (PBS), which was subjected tothree freeze-thaw cycles under vacuum, followed by pelleting of celldebris by centrifugation, and storage of the supernatant at −80° C.

The concentration of thiol in the suspension of damaged CHO cells wasmeasured as 4 mM using DTNB ((5,5′-dithiobis(2-nitrobenzoic acid);Ellman's reagent). Humanized anti-folate receptor 1 (FOLR1) IgG1 (1 mg)was immobilized on 0.05 ml of Protein A bead (RepliGen) and then treatedwith pre-mixed 0.5 ml PBS and 0.1 ml damaged CHO cell supernatant. Theconcentration of thiol in this mixture was 0.67 mM.

The sample was rotated for about 2.2 hours at ambient temperature,following which the supernatant was removed and the beads were washedwith PBS four times (1.5 ml each). The thiol content of the immobilizedantibody on the beads was then analyzed by the addition of 0.6 ml of PBScontaining 0.5 mM DTNB, rotation for about 10 minutes, centrifugation,and measurement of the absorbance of the supernatant at 412 nm.

The number of thiol residues per antibody molecule after incubation withdamaged CHO cell supernatant was calculated as 4.2 thiol/antibody. Asimilar value of thiol/antibody was measured using another humanizedIgG1 antibody, huN901, incubated with the damaged CHO cell supernatant,suggesting that the mechanism for the reduction of disulfide bonds inIgG1 molecules by the damaged CHO cell supernatant is general and notspecific to a particular IgG1.

Methylene blue was then tested at different concentrations to determinewhether it could protect against disulfide bond reduction during theincubation of antibody with damaged CHO cell supernatant. FIG. 1 is agraph showing the results of immobilized 1 mg humanized IgG1 withpre-mixed 0.5 ml PBS and 0.1 ml damaged CHO cell supernatant in thepresence of methylene blue, rotation for 2.2 hours at ambienttemperature, and a similar thiol analysis of immobilized antibody asabove. Unexpectedly, very low concentrations of methylene blue (0.025and 0.05 mM) protected against the reduction of antibody disulfidebonds. Surprisingly, these concentrations were significantly lower thanthat of thiol (0.67 mM) in the mixture of PBS and damaged CHO cellsupernatant.

As shown in FIG. 1, only 0.38 and 0.33 thiol per antibody were detectedin the antibody samples containing mixtures of damaged CHO cellsupernatant with methylene blue (0.025 mM and 0.05 mM respectively)after 2.2 hours of incubation, in contrast to the 4.2 thiol per antibodydetected in the antibody sample containing damaged CHO supernatant only(without methylene blue) after 2.2 hours of incubation.

In a similar experiment, a sample containing 1 mg immobilized antibodywas incubated with a mixture of damaged CHO cell supernatant and 0.05 mMmethylene blue for 2.2 hours, followed by washing of beads as above,after which the antibody was eluted from Protein A bead using 100 mMacetic acid containing 150 mM NaCl, neutralized to pH 7 by addition of1.25 M KH₂PO₄ solution. Using an aliquot of the neutralized elutedantibody sample, the thiol per antibody was measured as 0.002 thiol perantibody using DTNB, thus indicating the protection against antibodydisulfide reduction by methylene blue.

In addition to the finding that methylene blue protects against antibodydisulfide reduction, it was unexpectedly found that methylene blueoffered protection from antibody disulfide reduction at lower,sub-stoichiometric concentrations (0.025 mM and 0.05 mM) compared to theconcentration of total thiol in the CHO supernatant/PBS mixture (0.67mM).

It is also useful that the anti-reduction agent added to protect thedisulfide bonds in antibody from reduction by the reducing proteins inthe damaged cell supernatant does not covalently modify the antibody atthiol residues. Covalent modification of thiol residues derived fromreduction of native inter-chain disulfides could increase theimmunogenicity of antibody and could also adversely affect thephysicochemical behavior of the antibody.

To assess if there was any covalent modification of the antibody uponaddition of methylene blue, the absorbance of methylene blue-treatedanti-FOLR1 IgG1 was monitored at the absorbance maxima of methylene blue(610 nm). The molecular weight of the antibody was assessed by massspectrometry. The eluted neutralized antibody sample described above(following methylene blue treatment) did not show blue color and had anabsorbance of 0.000 at 610 nm, which is the absorbance maxima formethylene blue (extinction coefficient, 610 nm=8800 M-1 cm-1),suggesting that methylene blue did not covalently modify the antibody.

Another aliquot of the eluted neutralized antibody was deglycosylatedand analyzed by mass spectrometry. As shown in FIG. 2, the antibodysample that was treated with 0.05 mM methylene blue showed a major masspeak at 145694, which was similar to that of the untreated antibody,suggesting that methylene blue treatment did not lead to themodification of the antibody.

Example 2: Coenzyme Q Analogs Decreased Antibody Disulfide-Reduction

Coenzyme Q analogs were tested at different concentrations to determinewhether they had a protective effect on disulfide reduction during theincubation of antibody with damaged CHO cell supernatant (FIG. 3).Humanized N901 (Anti-CD56 IgG1; 1 mg) was immobilized on 0.05 ml ofProtein A bead (RepliGen) and then treated with pre-mixed 0.4 ml PBS and0.1 ml damaged CHO cell supernatant (described in Example 1) with orwithout the addition of coenzyme Q analogs. The concentration of thiolin these mixtures was 0.8 mM.

The samples were rotated for about 1.5 hours at ambient temperature,following which the supernatant was removed and the beads washed withPBS three times. The thiol content of the immobilized antibody on thebeads was then analyzed by the addition of 0.6 ml of mixture of PBS and0.5 mM DTNB, rotation for about 10 minutes, centrifugation, andmeasurement of the absorbance of the supernatant at 412 nm. The numberof thiol residues per antibody molecule after incubation with damagedCHO cell supernatant (without any coenzyme Q analog) was measured as 6.4thiol/antibody.

In contrast, as shown in FIG. 3, the co-incubation of antibody anddamaged CHO supernatant with coenzyme Q0(2,3-dimethoxy-5-methyl-p-benzoquinone) resulted in a significantprotection from antibody disulfide reduction, with only 0.34 and 0.63thiol per antibody at 0.2 mM and 0.1 mM coenzyme Q0 respectively.

Coenzyme Q0 is known to react with thiols in a stoichiometric manner (W.Li, J. Heinze, and W. Haehnel, J. Am. Chem. Soc., 127, 6140-6141, 2005).It was highly unexpected, however, to observe that coenzyme Q0 waseffective in lowering the antibody disulfide reduction atsub-stoichiometric molar concentrations (0.2 and 0.1 mM) compared to thetotal thiol concentration in the damaged CHO cell supernatant mixture(0.8 mM).

Also, as shown in FIG. 3, coenzyme Q2(2,3-dimethoxy-5-methyl-6-geranyl-p-benzoquinone), decreased antibodydisulfide-reduction by damaged CHO supernatant at low concentrations(0.2 and 0.1 mM) that were sub-stoichiometric in molar terms compared tothe total thiol concentration in the damaged CHO cell supernatantmixture (0.8 mM).

In contrast, coenzyme Q10 did not show significant protection towardantibody-disulfide reduction by damaged CHO supernatant when added at0.2 and 0.1 mM concentrations. It is possible that the low solubility ofcoenzyme Q10 hinders its ability to protect the antibody-disulfide fromreduction.

A sample of humanized N901 antibody incubated with coenzyme Q0 wasanalyzed by mass spectrometry and showed a mass of 146148, which wassimilar to that of control, unreduced humanized N901 antibody (mass146150), thus showing that the antibody was not covalently modified bycoenzyme Q0.

Example 3: 1,2-naphthoquinone-4-sulfonic Acid Decreased AntibodyReduction

The effect of 1,2-naphthoquinone-4-sulfonic acid (NQS) onantibody-disulfide reduction was studied by incubating1,2-naphthoquinone-4-sulfonic acid at several concentrations withantibody and damaged CHO lysate. The damaged CHO cell lysate wasgenerated by the addition of 0.5 ml RIPA buffer (50 mM Tris-HCl, pH 7.4,1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate) to 100million CHO cells. The thiol concentration in CHO cell lysate wasmeasured by Ellman's assay as 2.1 mM.

Each sample contained pre-mixed 0.5 ml RIPA buffer (50 mM Tris-HCl, pH7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate)with 1 mg humanized N901 antibody, with or without1,2-naphthoquinone-4-sulfonic acid (25 micromolar, or 50 micromolar, or100 micromolar), which was added to a cell pellet of 100 million CHOcells, and the resuspended cells were incubated at ambient temperaturefor 30 minutes, following which the cell debris were pelleted bycentrifugation (16,000 g, 5 min). The supernatant from each sample wasadded to 50 microliter immobilized-Protein A beads (RepliGen) androtated at ambient temperature for 3 hours, after which the supernatantwas removed and the beads washed with PBS.

The thiol content of the immobilized antibody was determined by theaddition of 0.6 ml of PBS containing 0.5 mM DTNB (Ellman's reagent) tothe beads, rotation for about 10 minutes, centrifugation, and absorbancemeasurement of supernatant at 412 nm. As shown in FIG. 4, the controlantibody sample (without 1,2-naphthoquinone-4-sulfonic acid) uponreduction by CHO cell lysate showed a thiol/antibody ratio of 5.74.

In contrast, addition of low concentrations of naphthoquinone-4-sulfonicacid (NQS) to the mixture of antibody sample and CHO lysatesignificantly decreased the thiol/antibody ratio. The thiol/antibodyratio was 3.4, 2.2, and 1.25 upon addition of 0.025 mM, 0.05, and 0.1 mMnaphthoquinone-4-sulfonic acid (NQS), respectively. The concentrations(0.025-0.1 mM) of NQS that were effective at decreasing the extent ofantibody reduction caused by CHO lysate were surprisingly much lowerthan the thiol concentration in the CHO lysate (2.1 mM).

Example 4: Anthraquinone-2-sulfonic Acid Decreased Antibody-DisulfideReduction

The effect of anthraquinone-2-sulfonic acid (AQS) on antibody-disulfidereduction was studied during co-incubation of anthraquinone-2-sulfonicacid with antibody and damaged CHO lysate. Each sample containedpre-mixed 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5%sodium deoxycholate, 0.1% sodium dodecylsulfate) with 1 mg humanizedN901 antibody, with or without 0.2 mM anthraquinone-2-sulfonic acid(AQS), which was added to a cell pellet of 100 million CHO cells, andthe resuspended cells were incubated at ambient temperature for 30minutes, following which the cell debris were pelleted by centrifugation(16,000 g, 5 min). The thiol concentration in CHO cell lysate was 1 mM(Ellman's assay).

The supernatant from each sample was added to 50 microliterimmobilized-Protein A beads (RepliGen) and rotated at ambienttemperature for 3 hours, after which the supernatant was removed and thebeads washed with PBS. The thiol content of the immobilized antibody wasdetermined by the addition of 0.6 ml of PBS containing 0.5 mM DTNB(Ellman's reagent) to the beads, rotation for about 10 minutes,centrifugation, and absorbance measurement of supernatant at 412 nm. Asshown in FIG. 5, the control antibody sample (without anyanthraquinone-2-sulfonic acid) incubated with CHO cell lysate for 3hours underwent reduction of antibody-disulfide resulting in 4.86thiol/antibody.

In contrast, the antibody sample (with 0.2 mM anthraquinone-2-sulfonicacid) incubated with CHO cell lysate for 3 hours showed significantlyless disulfide-reduction resulting in 2.46 thiol/antibody (49% decreasein antibody-disulfide reduction).

In another experiment, 1 mg huN901 antibody was incubated with CHOlysate, with or without 0.4 mM anthraquinone-2-sulfonic acid for 4hours. The mixture was then analyzed for thiol/antibody as above. Inthis experiment, the control antibody sample (without anyanthraquinone-2-sulfonic acid) following incubation with CHO cell lysatefor 4 hours showed 4.34 thiol/antibody.

In contrast, the antibody sample (with 0.4 mM anthraquinone-2-sulfonicacid) following incubation with CHO cell lysate for 4 hours showedsignificantly less disulfide-reduction, resulting in a value of 1.65thiol/antibody (62% decrease in antibody-disulfide reduction).

In a separate experiment, the reactivity of anthraquinone-2-sulfonicacid (AQS) with a model thiol (L-cysteine ethyl ester) at ambienttemperature was investigated by incubating 1 mM AQS with 1 mM cysteineethyl ester at ambient temperature in PBS, using Ellman's assay of thiolgroups to measure the extent of reaction. At time points of 1 hour, 2hours, 3 hours, and 4 hours, the percent decrease in thiol values forthe 1 mM AQS with 1 mM cysteine ethyl ester mixture versus thiol in a 1mM cysteine ethyl ester (No AQS) control were 2%, 8%, 12%, and 20%,respectively.

It was surprising to observe a 49% decrease in antibody-disulfidereduction by CHO lysate following 3 hours incubation with 0.2 mM AQS,and a 62% decrease in antibody-disulfide reduction by CHO lysatefollowing 4 hours incubation with 0.4 mM AQS. The extent of protectionagainst antibody disulfide-reduction was much greater than expectedgiven the thiol reaction kinetics observed at 3 hours (12% lower thiol)and 4 hours (20% lower thiol), which were based on the reaction of 1 mMcysteine ethyl ester with 1 mM AQS.

The effect of incubating cells with anthraquinone-2-sulfonic acid (AQS)was tested using Calu3 human lung cancer cells. Effects on cellviability were assayed by microscopic observation of cells and byATP-based viability assay using Cell Titer Glo reagent (Promega). Uponincubation of Calu3 cells with 0.25 and 0.5 mM AQS for 1 day, no visiblecytotoxic effects were observed and the luminescence values obtainedusing Cell Titer Glo reagent were similar (100% and 85%, respectively)to that of control cells without AQS, demonstrating that AQS is notcytotoxic at these concentrations.

In another experiment to test the effect of AQS on the viability ofcells, CHO cells producing antibody were kept in continuous culture forabout two weeks, and then were treated with 0.25 mM, 0.5 mM, and 0.75 mMAQS for 1 day. Cell viability was measured after this treatment. Theviability count using trypan blue for control, untreated cells was63.8%, which was similar to that for AQS-treated cells (66.2%, 64.8%,and 61.7% for 0.25, 0.5, and 0.75 mM AQS-treated cells, respectively).AQS, therefore, was not cytotoxic to antibody-producing cells.

To test the effect of AQS-treatment on covalent modification ofantibody, humanized N901 IgG1 (2 mg in 1 ml PBS) was treated with 1 mMAQS and 0.35 ml CHO cell lysate (prepared by lysis and centrifugation of100 million CHO cells in 1 ml RIPA buffer), then added to 100 microliterProtein A beads, and rotated for 3 hours. The immobilized antibody waswashed with PBS, eluted in 100 mM acetic acid containing 150 mM NaCl,neutralized to pH 7 with 1.25 M KH₂PO₄ solution, and dialyzed. Thedialyzed antibody was deglycosylated and analyzed by mass spectrometry,which showed a mass of 146156 that was similar to that of control,unreduced antibody (mass 146154) (FIG. 6B), indicating that AQS did notcovalently modify the antibody (FIG. 6A).

Example 5: Lipoic Acid Decreased Antibody Reduction

Humanized anti-folate receptor-1 IgG1 (1 mg) was immobilized on 0.05 mlof Protein A beads (RepliGen) and then treated with pre-mixed 0.5 ml PBSand 0.1 ml damaged CHO cell supernatant with or without 0.5 mM and 2 mMlipoic acid (also named as 1,2-dithiolane-3-pentanoic acid; or6,8-dithiooctanoic acid; or DL-6,8-thioctic acid; added using a stocksolution of lipoic acid in DMSO), pre-incubated for 20 min beforeaddition to immobilized antibody. The concentration of total thiol(derived from damaged cells) in this mixture was 0.67 mM.

The samples were rotated for about 2.5 hours at ambient temperature,then centrifuged to pellet beads, following which the supernatants wereremoved and the beads washed with PBS four times. The thiol content ofthe immobilized antibody on the beads was then analyzed by the additionof 0.6 ml PBS containing 0.5 mM DTNB to the beads, which were incubatedwith rotation for about 5 minutes and centrifuged to pellet the beats.The absorbance of the supernatant was measured at 412 nm.

In the control antibody sample without lipoic acid, the number of thiolresidues per antibody molecule after incubation with damaged CHO cellsupernatant was calculated as 5.5. For the 0.5 mM and 2 mM lipoicacid-treated mixtures of antibody and damaged CHO cell supernatant, thethiol/antibody ratios were 1.88 and 1.08, respectively (FIG. 7). Even ata lipoic acid concentration of 0.5 mM, which was lower than the totalthiol concentration of 0.67 mM, a significant decrease of thiol/Ab wasobtained for the mixture of damaged CHO cell supernatant with antibody.

In another experiment, the viability of CHO cells treated with 0.75 mMlipoic acid for 1 day was measured as 98.1%, which was similar to theviability of control, untreated cells (98.9% viability). Lipoic acidtreatment, therefore, was not cytotoxic to CHO cells.

To test the effect of lipoic acid-treatment on covalent modification ofantibody, humanized N901 IgG1 (2 mg in 1 ml PBS) was treated with 1 mMlipoic acid and 0.35 ml CHO cell lysate (prepared by lysis andcentrifugation of 100 million CHO cells in 1 ml RIPA buffer), then addedto 100 microliter Protein A beads, and rotated for 3 hours. Theimmobilized antibody on beads was washed with PBS, eluted in 100 mMacetic acid containing 150 mM NaCl, neutralized to pH 7 with 1.25 MKH₂PO₄ solution, and dialyzed. The dialyzed antibody was deglycosylatedand analyzed by mass spectrometry, which showed a mass of 146152 thatwas similar to that of control, unreduced antibody (mass 146154) (FIG.8B), indicating that lipoic acid did not covalently modify the antibody(FIG. 8A).

Lipoic acid contains a strained 5-membered cyclic disulfide (S. Sunner,Nature, 176, 217, 1955). The strained cyclic disulfide group in lipoicacid is more reactive toward thiol than non-cyclic disulfides, whichwould favor the reduction of lipoic acid by CHO cell thiol proteins incomparison to antibody disulfide bonds.

However, the reduced lipoic acid is a 1,3-dithiol, which has a higherreduction potential than monothiols (W. J. Lees and G. M. Whitesides, J.Org. Chem., 58, 642-647, 1993), and it is possible that reduced lipoicacid (dihydrolipoic acid) could reduce antibody disulfide bonds. It wastherefore unexpected that lipoic acid decreased antibody disulfidereduction by damaged CHO cell suspension or lysate.

Example 6: Cystine Dimethyl Ester and Cystine Diethyl Ester EachDecreased Antibody Reduction

The effect of L-cystine dimethyl ester (CDME) on antibody-disulfidereduction was studied following co-incubation of CDME at severalconcentrations with antibody and damaged CHO lysate. Each samplecontained pre-mixed 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40,0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate) with 1 mghumanized N901 antibody, with or without 0.05-0.5 mM L-cystine dimethylester (CDME), which was added to a cell pellet of 100 million CHO cells,and the resuspended cells were incubated at ambient temperature for 30minutes, following which the cell debris were pelleted by centrifugation(16,000 g, 5 min). The thiol concentration in CHO cell lysate was 1 mM(Ellman's assay).

The supernatant from each sample was added to 50 microliterimmobilized-Protein A beads (RepliGen) and rotated at ambienttemperature for 3 hours, after which the supernatant was removed and thebeads washed with PBS. The thiol content of the immobilized antibody wasdetermined by the addition of 0.6 ml of PBS containing 0.5 mM DTNB(Ellman's reagent) to the beads, rotation for about 10 minutes,centrifugation, and absorbance measurement of the supernatant at 412 nm.

The control, antibody sample (without any CDME) showed 5.20thiol/antibody generated by reduction of antibody disulfide by CHOlysate. In contrast, as shown in FIG. 9, treatment with 0.05 mM, 0.1 mM,0.2 mM, and 0.5 mM CDME significant decreased antibody-disulfidereduction by CHO lysate, resulting in 2.75, 1.85, 1.56, and 1.19thiol/antibody, respectively. Unexpectedly, CDME decreased thedisulfide-reduction in antibody by CHO lysate, even at lowconcentrations of CDME (0.05-0.5 mM) that were significantly lower thanthe total thiol concentration in CHO lysate mixture (1 mM).

In another experiment, cystine dimethyl ester (CDME) and cystine diethylester (CDEE) were added at 0.75 mM each to a mixture of 2 mg humanizedN901 IgG1 and CHO lysate, and the samples were rotated at 4° C.overnight, after which the thiol/antibody was determined using PBS andDTNB mixture as described above. As shown in FIG. 10, addition of 0.75mM of CDME or CDEE decreased the extent of disulfide reduction ofantibody caused by CHO lysate to 0.96 and 1.02 thiol/antibody,respectively, compared to the thiol/antibody value of 2.29 for thecontrol sample without any CDME or CDEE. FIG. 11 shows that incubationof antibody with a combination of anthraquinone-2-sulfonic acid (AQS)and CDME at 0.2 mM each or 0.4 mM each decreased the extent ofantibody-disulfide reduction by CHO lysate significantly.

In contrast to the control sample (without AQS or CDME) that showed 4.34thiol/antibody, treatment with a combination of AQS and CDME decreasedthe antibody-disulfide reduction to 1.12 thiol/antibody, both for 0.2 mMeach, and 0.4 mM each of AQS and CDME (FIG. 11). These values ofthiol/antibody obtained with the combination of AQS and CDME were lowerthan those with 0.4 mM AQS alone (1.65 thiol/antibody) or with 0.4 mMCDME alone (1.37 thiol/antibody), indicating that the combinations wereeven more effective in decreasing antibody reduction than the individualagents.

FIG. 12A and FIG. 12B show the results of an experiment in which a CHOcell culture producing recombinant humanized IgG1 antibody was subjectedto depth filtration and the resulting harvest cell culture fluid (HCCF)was frozen at −80° C. After thaw, two samples were prepared which werekept under nitrogen in bottles at ambient temperature. To one sample wasadded a combination of 0.5 mM anthraquinone-2-sulfonic acid (AQS) and0.5 mM cystine dimethyl ester dihydrochloride (CDME), termed “AQC”.

Another sample, without any added AQS or CDME, served as the control. Atvarious time points, samples were quenched with 5 mM N-ethylmaleimide(NEM), purified using immobilized Protein A, and subjected tonon-reducing SDS-Protein Lab Chip electrophoretic analysis.

FIG. 15A shows the non-reducing SDS-Protein Lab Chip electrophoreticdata, which are quantitatively shown in FIG. 15B. The combination of AQSand CDME (“AQC”) significantly decreased antibody fragmentation at alltime points from 2 hours to 68 hours compared to the control without anyAQS or CDME added (FIG. 15A, 15B). For example, at 24 hours the controlsample (without any AQS or CDME) showed 89.6% fragmentation of antibody,whereas the sample treated with the combination of AQS and CDME (“AQC”)showed only 5% fragmentation, which was similar to that of untreatedantibody.

A significant decrease in the percentage fragmentation (andcorrespondingly higher percentage of intact antibody) was seen at alltime points analyzed for the sample treated with the combination of AQSand CDME compared to the control sample without any AQS or CDME (FIG.15B).

To test the effect of CDME-treatment on covalent modification ofantibody, humanized N901 IgG1 (2 mg in 1 ml PBS) was treated with 1 mMCDME and 0.35 ml CHO cell lysate (prepared by lysis and centrifugationof 100 million CHO cells in 1 ml RIPA buffer), then added to 100microliter Protein A beads, and rotated for 3 hours. The immobilizedantibody was washed with PBS, eluted in 100 mM acetic acid containing150 mM NaCl, neutralized to pH 7 with 1.25 M KH₂PO₄ solution, anddialyzed. The dialyzed antibody was deglycosylated and analyzed by massspectrometry, which showed a mass of 146150 that was similar to that ofuntreated antibody (mass 146154) (FIG. 13B), indicating that cystinedimethyl ester treatment did not covalently modify the antibody (FIG.13A).

The aqueous solubilities of L-cystine dimethyl ester dihydrochloride andL-cystine diethyl ester dihydrochloride were compared to those ofL-cystine and L-cystine dihydrochloride. Unexpectedly, the solubilitiesof L-cystine dimethyl ester dihydrochloride and L-cystine diethyl esterdihydrochloride in water were found to be much higher than those ofL-cystine and L-cystine dihydrochloride.

In contrast to L-cystine, which could be dissolved in water at onlyabout 0.5 mM, the L-cystine dimethyl ester dihydrochloride and L-cystinediethyl ester dihydrochloride could be dissolved in water even at 1000mM. The aqueous solubility of L-cystine dimethyl ester dihydrochlorideand L-cystine diethyl ester dihydrochloride, therefore, was 2000 timeshigher than that of L-cystine.

For addition of concentrated solutions of L-cystine dimethyl esterdihydrochloride and L-cystine diethyl ester dihydrochloride to media atpH 7, it would be desirable to add neutralized stock solutions at aboutpH 7. The aqueous solubility of L-cystine dimethyl ester dihydrochlorideand L-cystine diethyl ester dihydrochloride were investigated afterneutralization to about pH 7 by addition of IN NaOH.

Unexpectedly, it was found that even after neutralization to about pH 7,the L-cystine dimethyl ester dihydrochloride and L-cystine diethyl esterdihydrochloride samples remained soluble at concentrations higher than100 mM. The unexpectedly high aqueous solubilities of L-cystine dimethylester dihydrochloride (CDME) and L-cystine diethyl ester dihydrochloride(CDEE) after neutralization to about pH 7 allow the addition ofconcentrated solutions of these agents (CDME, or CDEE) to cell culturemedium to obtain final concentrations in mM range without altering thepH of the medium.

If desired, concentrated solutions of L-cystine dimethyl esterdihydrochloride or L-cystine diethyl ester dihydrochloride and base(such as NaOH) can also be added to the medium without altering the pHof the medium.

The disulfide-reduction protection offered by a saturated solution ofL-cystine dihydrochloride was compared with a L-cystine dimethyl ester(L-CDME) dihydrochloride solution. The saturated solution of L-cystinedihydrochloride was prepared by suspending 11.7 mg of cystinedihydrochloride (FW 313.2) in 7.47 ml of 50 mM potassium phosphatebuffer, pH 7. The pH was adjusted to pH 7.1 and the sample was rotatedovernight at ambient temperature followed by centrifugation to removeundissolved L-cystine dihydrochloride. This saturated solution ofL-cystine was at a concentration less than 5 mM, which would have beenthe theoretical concentration if all of the initially added 11.7 mgL-cystine dihydrochloride had dissolved completely in 7.47 ml. Incontrast, a fully soluble 5 mM L-cystine dimethyl ester dihydrochloridesolution could be readily prepared in 50 mM potassium phosphate buffer,pH 7, and its pH was adjusted to pH 7.1. L-cystine dimethyl ester issoluble at much higher concentrations; the concentration of 5 mM used inthis experiment was for comparison with L-cystine.

Humanized FOLR1 IgG1 antibody (3 mg) in 0.3 ml of 50 mM potasssiumphosphate buffer, pH 7, was incubated with 0.59 ml of above saturatedL-cystine solution, or with 0.59 ml of 5 mM L-CDME solution, or with0.59 ml of 50 mM potasssium phosphate buffer, pH 7. Lysates of CHO cells(˜60 million cells lysed in 0.6 ml RIPA buffer and clarified bycentrifugation) were added to each of these mixtures. The pH values ofall samples were adjusted to 7. Each sample was at a final volume of 1.5ml, which contained 2 mg/ml humanized antibody, without any protectant(control), or with L-cystine (derived from saturated L-cystinesolution), or with 2 mM L-CDME. The samples were incubated with 200microliter immobilized-Protein A beads (RepliGen) and rotated at ambienttemperature for 3 hours, after which the supernatant was removed and thebeads washed with PBS. The thiol content of the immobilized antibody wasdetermined by the addition of 0.2 ml of PBS containing 0.5 mM DTNB(Ellman's reagent) to the beads, rotation for about 10 minutes,centrifugation, and absorbance measurement of the supernatant at 412 nm.As shown in FIG. 14, the control sample showed reduction of disulfidebonds in antibody resulting in 1.96 thiol per antibody. The samplederived from saturated L-cystine dihydrochloride showed a lower extentof antibody-disulfide reduction, with about 0.74 thiol per antibody. Itwas highly surprising and unexpected that 2 mM L-CDME was superior toL-cystine toward antibody-disulfide reduction, resulting in only about0.25 thiol per antibody.

Humanized FOLR1 IgG1 antibody (2 mg/ml) in 50 mM potassium phosphatebuffer, pH 7, was incubated with 2 mM CDME, or 2 mM AQS, or a mixture of1 mM CDME and 1 mM AQS, followed by addition of lysate of CHO cells (˜40million cells lysed in 0.4 ml RIPA buffer and clarified bycentrifugation). The pH values of all samples were adjusted to 7. Thesamples were incubated with 200 microliter immobilized-Protein A beads(RepliGen) and rotated at ambient temperature for 3 hours, after whichthe supernatant was removed and the beads washed with PBS. The thiolcontent of the immobilized antibody was determined by the addition of0.2 ml of PBS containing 0.5 mM DTNB (Ellman's reagent) to the beads,rotation for about 10 minutes, centrifugation, and absorbancemeasurement of the supernatant at 412 nm. As shown in FIG. 15, thecontrol sample showed reduction of disulfide bonds in antibody resultingin 0.95 thiol per antibody. The 2 mM AQS containing sample showed alower extent of antibody-disulfide reduction, with about 0.18 thiol perantibody. The 2 mM CDME and the 1 mM CDME+1 mM AQS combination treatedsamples showed much lower levels of Ab-disulfide reduction, resulting inonly about 0.02 and 0.03 thiol per antibody, respectively.

In another experiment, a 14 day harvest cell culture fluid of humanizedFOLR1 IgG1 producing CHO cells was treated with 20% (v/v) ofmicrofluidized CHO cells in the absence or presence of additives (1 mMCDME, 1 mM AQS, or 1 mM CDME+1 mM AQS, termed “AQC”). Themicrofluidization was carried out using 3 liter CHO cells from abioreactor, which were resuspended into 300 ml PBS, processed throughmicrofluidizer, centrifuged and filtered through 0.22 micrometermembrane. The samples of HCCF+microfluidized CHO cells (20% v/v),without or with CDME, AQS, or CDME+AQS, were incubated for 6 h. Thesamples were quenched with 5 mM N-ethylmaleimide (NEM), purified usingimmobilized Protein A, and subjected to non-reducing SDS-Protein LabChip electrophoretic analysis. As shown in FIG. 16, 78% fragmentationwas observed for the no additive control. In contrast, much lowerfragmentations were observed for samples which contained 1 mM CDME, 1 mMAQS, and 1 mM CDME and 1 mM AQS (termed “AQC”).

In another experiment, the viability of CHO cells treated with 0.75 mML-cystine dimethyl ester (CDME) for 1 day was measured as 98.9%, whichwas similar to the viability of control, untreated cells (98.9%viability). L-cystine dimethyl ester (CDME) treatment, therefore, wasnot cytotoxic to CHO cells.

To test the effect of L-cystine bis(t-butyl ester) (CDBE) on protectingantibody disulfide bonds from reduction by CHO lysate, humanized FOLR1IgG1 antibody (2 mg) was incubated with CHO lysate in RIPA buffer (0.6ml lysate of 60 million CHO cells in 0.6 ml RIPA buffer, clarified bycentrifugation) without or with 0.5 or 1 mM CDBE. The samples wererotated at ambient temperature with 200 microliter immobilized-Protein Abeads (RepliGen) for 2 hours, after which the supernatant was removedand the beads washed with PBS. The thiol content of the immobilizedantibody was determined by the addition of 0.2 ml of PBS containing 0.5mM DTNB (Ellman's reagent) to the beads, centrifugation, and absorbancemeasurement of the supernatant at 412 nm. As shown in FIG. 17, thecontrol sample (without any CDBE) showed reduction of disulfide bonds inantibody resulting in 2.26 thiol per antibody. The 0.5 mM CDBE and the 1mM CDBE treated samples showed much lower levels of Ab-disulfidereduction, resulting in only about 0.16 and 0.10 thiol per antibody,respectively (that is, about 93% and 96% decrease in disulfide reductioncompared to the control without CDBE, respectively).

In another experiment, the use of 0.25 and 2 mM CDBE during theincubation of antibody with CHO cell lysate showed about 78% and 98%decrease in disulfide reduction compared to the control without CDBE,respectively.

The solubility of L-cystine bis(t-butyl ester) (CDBE) was explored bydissolving L-cystine bis(t-butyl ester) dihydrochloride in water.Unexpectedly, L-cystine bis(t-butyl ester) dihydrochloride was found tobe soluble in water even at a high concentration of 500 mM.

To test the effect of Di-N-acetyl L-cystine on protecting antibodydisulfide bonds from reduction by CHO lysate, humanized FOLR1 IgG1antibody (3 mg) was incubated with CHO lysate (0.6 ml lysate of 60million CHO cells in 0.6 ml RIPA buffer, clarified by centrifugation) in100 mM phosphate buffer (total volume 1.5 ml; pH 7), without any addedcompound or with 5 mM Di-N-acetyl L-cystine or with 2 mM L-cystinedimethyl ester. The samples were rotated at ambient temperature with 200microliter immobilized-Protein A beads (RepliGen) for 2 hours, afterwhich the supernatant was removed and the beads washed with PBS. Thethiol content of the immobilized antibody was determined by the additionof 0.2 ml of PBS containing 0.5 mM DTNB (Ellman's reagent) to the beads,centrifugation, and absorbance measurement of the supernatant at 412 nm.The control sample (without any added compound) showed reduction ofdisulfide bonds in antibody resulting in 1.16 thiol per antibody. The 5mM Di-N-acetyl L-cystine and 2 mM L-cystine dimethyl ester treatedsamples showed significantly lower levels of Ab-disulfide reduction,resulting in only about 0.42 and 0.13 thiol per antibody, respectively(that is, about 64% and 89% decrease in disulfide reduction compared tothe control without any added compound, respectively).

Example 7: Oxidized Glutathione (GSSG) Alone or in Combination withGlutathione Reductase Decreased Antibody Reduction

The effect of a combination of glutathione reductase and oxidizedglutathione (GSSG) on antibody-disulfide reduction was studied byco-incubating antibody and CHO cell lysate. Each sample contained 1 mlpre-mixed RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% sodium dodecylsulfate) with 1 mg humanized N901antibody, with or without 2 mM GSSG alone or a combination of 2 mM GSSGand 1.5 units (15 microgram) glutathione reductase (purified fromBaker's yeast). Each sample was added to a cell pellet of 100 millionCHO cells, and the resuspended cells were incubated at ambienttemperature for 30 minutes, following which the cell debris werepelleted by centrifugation (16,000 g, 5 min). The thiol concentration inCHO cell lysate was 1 mM (Ellman's assay).

The supernatant from each sample was added to 50 microliterimmobilized-Protein A beads (RepliGen) and rotated at ambienttemperature for 2.5 hours, after which the supernatant was removed andthe beads washed with PBS. The thiol content of the immobilized antibodywas determined by the addition of 0.6 ml of PBS containing 0.5 mM DTNB(Ellman's reagent) to the beads, rotation for about 10 minutes,centrifugation, and absorbance measurement of the supernatant at 412 nm.The control, antibody sample (without any GSSG or glutathione reductaseadded) showed 5.63 thiol/antibody generated by reduction of antibodydisulfide by CHO lysate.

As shown in FIG. 18, treatment with GSSG alone decreasedantibody-disulfide reduction by CHO lysate, resulting in 0.98thiol/antibody. The combination of GSSG and glutathione reductaselowered the thiol/antibody ratio further to 0.63. The combination ofGSSG and glutathione reductase, therefore, was even more effective thanGSSG alone in decreasing the reduction of antibody disulfide bondsinduced by CHO cell lysate.

Example 8: Disulfiram Decreased Antibody Reduction

Humanized N901 IgG1 (1 mg) was immobilized on 0.05 ml of Protein A bead(RepliGen) and treated with pre-mixed 0.45 ml PBS and 0.05 ml damagedCHO cell supernatant with or without 0.1 mM and 0.2 mM disulfiram (alsonamed as tetraethylthiuram disulfide; added using a stock solution ofdisulfiram in DMSO). The concentration of total thiol (derived fromdamaged cells) in this mixture was 0.4 mM.

The samples were rotated for about 2.5 hours at ambient temperature,then centrifuged to pellet beads, following which the supernatants wereremoved and the beads washed with PBS four times. The thiol content ofthe immobilized antibody on the beads was then analyzed by the additionof 0.6 ml PBS containing 0.5 mM DTNB to the beads, rotation for about 30minutes, centrifugation, and measurement of the absorbance of thesupernatant at 412 nm.

In the control antibody sample without disulfiram, the number of thiolresidues per antibody molecule after incubation with damaged CHO cellsupernatant was calculated as 1.99. For the 0.1 mM and 0.2 mMdisulfiram-treated mixtures of antibody and damaged CHO cellsupernatant, the thiol/antibody ratios were 0.14 and 0.03, respectively(FIG. 19).

The effective concentrations of disulfiram (0.1 and 0.2 mM) thatdecreased antibody-disulfide reduction induced by damaged CHO cellsupernatant were unexpectedly lower than the total thiol concentrationof damaged CHO cell supernatant.

Example 9: Sparging with O₂ to Obtain Optimal Culturing Concentrations

The purpose of this experiment is to investigate antibody disulfidereduction in centrifuge-harvested cell culture fluid (HCCF) when thecell culture fluid is maintained at different levels of dissolved oxygen(DO) ranging from 0% to 150% air saturation. These levels of dissolvedoxygen are achieved by sparging the harvested cell culture fluid with amixture of nitrogen and oxygen gas. For each condition, 1 L ofcontinuously centrifuged HCCF is held in a 5 L bioreactor at ambienttemperature with moderate agitation (100 rpm), and a constant flow rateof nitrogen of 100 ml/min, and a dissolved oxygen set point of 0%, 50%,100%, or 150% air saturation, which is controlled by the addition ofoxygen gas. Samples are drawn periodically and analyzed for antibodydisulfide reduction using a Non-Reduced GelChip.

Example 10: Protection of Reduction of Disulfide Bonds in Antibody UsingCombinations of Air and CDME

A CHO cell culture producing recombinant humanized IgG1 antibody wassubjected to centrifugation and the resulting harvest cell culture fluid(HCCF) was frozen at −80° C. After thaw, samples were prepared, whichwere kept under nitrogen, or air, or with combinations of air and CDME(1 mM), or with combinations of nitrogen and CDME (1 mM, or 2 mM). Atvarious time points, samples were quenched with N-ethylmaleimide (NEM),purified using immobilized Protein A, and analyzed for fragments usingnon-reducing SDS-Protein Lab Chip electrophoresis. FIG. 20 shows thatthe control sample kept under nitrogen, which was initially about 48%fragmented, became nearly completely fragmented (99.4%) within 6.5 hoursand stayed reduced. In contrast, the sample overlayed with air becameless fragmented with time, reaching a plateau of about 25% fragmentationat 20 hours. Additionally, sparging with oxygen at various percentagesof dissolved O₂ showed protection against fragmentation whereas sampleskept under nitrogen alone had no protection against fragmentation. Theprotection against disulfide reduction is enhanced by the combination ofair overlay with CDME (even at low concentration of ˜1 mM), whichlowered the fragmentation further throughout the course of the study, upto 72 hours. Based on the levels of protection seen with air overlayalone and with oxygen sparging, the combination of oxygen sparging withCDME is also expected to be highly effective against antibody disulfidebond reduction. The combinations of CDME and nitrogen, were slightlyless protective than those with CDME and air; the combinations of 1 mMand 2 mM CDME with nitrogen protected the antibody from reduction up to20 hours, and 45 hours, respectively. Overall, the fragmentation withair and CDME together were significantly less than with nitrogen andCDME together. Therefore, CDME alone or a combination of oxygen spargingor air overlay with CDME is especially protective of the reduction ofdisulfide bonds of an antibody.

Example 11: Protection of Reduction of Disulfide Bonds of Humanized IgG4Antibody

A solution of humanized IgG4 in PBS (0.5 mg/ml; final concentration) wasincubated with 30% v/v CHO cell lysate (˜60 million cells lysed in 0.6ml RIPA buffer and clarified by centrifugation) with or without 1 mMCDME in a total volume of 2 ml. All samples were adjusted to pH 7 andincubated with 200 μl protein A beads for 4 hours while rotating at roomtemperature. After the incubation the supernatant was removed and beadswashed three times with 10 ml PBS. To analyze the thiol content of theprotein A-bound IgG4 antibody, the beads were incubated with a 0.5 mMDTNB solution for 5 min and the absorbance of the supernatant measuredat 412 nm. As shown in FIG. 21, the control sample showed reduction ofdisulfide bonds to an extent of 7.7 thiol groups per antibody. In thesample incubated with 1 mM CDME, the extent of reduction was much lower,resulting in only 0.004 thiol groups per antibody. For the antibodyincubated with 1 mM CDBE, the absorbance measured was zero; and thus noreduced disulfides were detected in these samples. Therefore, CDME andCDBE were efficient in protecting the disulfide bonds of an IgG4antibody from reduction by lysed CHO cells.

Example 12: Protection of Reduction of Disulfide Bonds of Humanized IgG2Antibody

Humanized IgG2 antibody (1 mg/ml; final concentration) was incubatedwith 40% v/v CHO cell lysate (˜40 million cells lysed with 0.4 ml RIPAbuffer and debris removed by centrifugation) in a total volume of 1 ml.The control sample was adjusted to pH 7 and rotated at RT for 5 hourswith 100 μl protein A beads. Additionally, IgG2 antibody was treated asdescribed for the control and contained either 1 mM CDME or 1 mM CDBE.After the incubation time of 5 hours, the supernatant was removed andthe beads were washed three times with 10 ml PBS before the thiol amountof the immobilized antibody was analyzed by adding 0.5 mM DTNB andmeasuring the absorbance at 412 nm. The control sample showed 0.84reduced thiols per antibody and samples incubated with 1 mM CDME or 1 mMCDBE showed near-zero reduction as shown in FIG. 22. Therefore, CDME andCDBE were efficient in protecting the disulfide bonds of IgG2 antibodyfrom reduction by lysed CHO cells.

Example 13: Protection of Reduction of Disulfide Bonds of Humanized IgG2Antibody by Using Zinc, Nitrate, Nickel, and Other Transition Elements

Purified M9346A antibody was adjusted to 5 mg/mL final concentration informulation buffer (25 mM histidine, 10% sucrose, 0.01% polysorbate-20,pH 6.3) with or without addition of 1 mM of a given media additive, andincreasing amounts of DTT were added. DTT concentration ranged from 0.34μM to 3.4 μM to 6.8 μM to 17 μM to 34 to 68 μM to 170 μM. Thecorresponding ratio of the M9346A antibody (at 5 mg/mL) to DTT rangedfrom 100 to 10 to 5 to 2 to 1 to 0.5 to 0.2. The mixtures were incubatedin a water bath at 37° C. for 1 h. Redox potential was measured using aredox probe. Free thiols were alkylated by addition of 10% (v/v) of 50mM N-ethyl-maleimide (NEM) solution. Samples were analyzed bynon-reduced sodium dodecyl sulfate denaturing capillary electrophoresis(NR-CE-SDS). As expected, control conditions without media additivesshowed a linear decline in redox potential when plotted against DTTconcentration on a logarithmic scale (FIG. 23, blue curve). Addition ofseveral additives led to modulation of the redox potential: severalcompounds, both organic and inorganic, showed constant redox potentialacross all DTT concentrations (examples include CDME, dichromate, andcopper).

Other substances modulated the redox potential in variable ways.Addition of cysteine led to constant redox potential at lowconcentrations of DTT, followed by a slow decrease at higher DTTconcentrations (FIG. 23, dark green curve). Other additives led tostep-wise modulation of redox potential, e.g. cystine.

When analyzing these samples for quantification of reduced species, wefound that numerous components protected antibodies from disulfidereduction over a wide span of DTT concentrations.

At 100 μM DTT, 80% of offered mAb remained intact in control condition(FIG. 24, blue curve). Numerous additives showed greater percentages ofintact antibody under the same conditions, spanning a window from80-100% intact antibody. For example, addition of 1 mM nickel sulfateyielded ˜90% intact antibody, and 1 mM copper sulfate, CDME, ordichromate were fully protective against disulfide reduction (100%intact antibody).

Example 14: Protection of Reduction of Disulfide Bonds of Humanized IgG2Antibody

Antibody reduction was induced in a biological assay by adding celllysate to purified monoclonal antibody. Purified M9346A antibody wasadjusted to 5 mg/mL final concentration in lysis buffer A (25 mM Tris,150 mM NaCl, 0.1% Tween-20, Complete® protease inhibitor cocktail, pH7.5) with or without addition of 1 mM of a given media additive.Increasing amounts of CHO cell lysate were added to mimic differentpercentages of cell lysis, ranging from 30-180%. Fresh NADPH was addedto a concentration of 400 μM. The mixtures were incubated in a waterbath at 37° C. for 1 h. Free thiols were alkylated by addition of 10%(v/v) of 50 mM N-ethyl-maleimide (NEM) solution. Samples were analyzedby non-reduced sodium dodecyl sulfate denaturing capillaryelectrophoresis (NR-CE-SDS). It was consistently found that 40% intactantibody was left after incubation with cell lysate to mimic 120% celllysis (FIG. 25, control condition). Addition of a specific inhibitor ofthioredoxin reductase at two different concentrations revealed thatantibody fragmentation is mediated by Thioredoxin Reductase, as nearly100% intact antibody was recovered after addition of inhibitor, but notsolvent alone (FIG. 25, DMSO control).

Using this assay, media additives were tested at 1 mM each. All of themprotected M9346A from disulfide reduction (FIG. 26). Eight of ninetested compounds protected M9346A nearly completely, only cysteineshowed partial protection, with ˜70% of intact antibody.

To test the capacity for protection further, the stringency of the assaywas increased. Antibody fragmentation began to saturate at lysateconcentrations mimicking greater than 120% cell lysis (FIG. 27, leftpanel). Therefore, the modeled percentage of lysis was kept constant at120% and increased the concentration of available NADPH, ranging from400 μM to 3200 μM (FIG. 27, middle panel). Antibody fragmentationsteeply increased through concentrations ranging from 400 μM to 1200 μM,but plateaued beyond 1200 μM. Therefore, NADPH concentrations were keptconstant at 1200 μM and incubation time was increased from 1 h to 3 h(FIG. 27, right panel).

Increasing assay stringency was achieved by modulating percentage ofmodeled cell lysis, NADPH concentration, and incubation time (stringencylevels 0-9), and multiple levels of antibody fragmentation weregenerated, covering the entire range from fully intact antibody tocompletely reduced antibody (FIG. 28, blue curve).

We found that 1 mM CDME, copper, or zinc were protective of disulfidereduction, even at conditions where the control without any additive was100% fragmented (FIG. 28).

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A method of inhibiting disulfide bond reduction in a recombinantprotein, antibody, or fragment thereof expressed in a host cell, themethod comprising adding an anti-reduction agent to a cell culturemedia, pre-harvest cell culture fluid, or harvest cell culture fluid,comprising the antibody or fragment thereof, wherein the anti-reductionagent is an organic or inorganic redox active substance.
 2. The methodof claim 1, wherein the anti-reduction agent is selected from the groupconsisting of ZnSO₄, CuSO₄, NiSO₄, NaNO₃, and any combinations thereof.3. A method of increasing production of a protein, antibody, or antibodyfragment with intact native disulfide bonds that is expressed in amammalian host cell, the method comprising adding an effective amount ofan anti-reduction agent to a cell culture media, pre-harvest cellculture fluid, or harvest cell culture fluid, comprising the antibody orfragment thereof, wherein the anti-reduction agent is an organic orinorganic redox active substance.
 4. The method of claim 3, wherein theanti-reduction agent is selected from the group consisting of ZnSO₄,CuSO₄, NiSO₄, NaNO₃, and any combinations thereof.
 5. The method ofclaim 3, wherein greater than about 80% of the protein, antibody, orantibody fragment produced has intact native disulfide bonds.
 6. Themethod of claim 5, wherein greater than about 90% of the protein,antibody, or antibody fragment produced has intact native disulfidebonds.
 7. The method of claim 1, wherein the method comprises adding theanti-reduction agent to the cell culture media.
 8. The method of claim7, wherein the method comprises adding the anti-reduction agent to thecell culture medium within about 48 hours of harvesting the cellculture.
 9. The method of claim 7, wherein the method comprises addingthe anti-reduction agent to the cell culture medium within about 24hours of harvesting the cell culture.
 10. The method of claim 7, whereinthe method comprises adding the anti-reduction agent to the cell culturemedium within about 12 hours of harvesting the cell culture.
 11. Themethod of claim 7, wherein the method comprises adding theanti-reduction agent to the cell culture medium within about 15 minutesof harvesting the cell culture.
 12. The method of claim 1, wherein themethod comprises adding the anti-reduction agent to the pre-harvest cellculture fluid.
 13. The method of claim 1, wherein the method comprisesadding the anti-reduction agent to the harvest cell culture fluid. 14.The method of claim 1, wherein the anti-reduction agent is added to afinal concentration in a range from about 0.01 mM to about 100 mM. 15.The method of claim 1, wherein the mammalian host cell is a ChineseHamster Ovary (CHO) cell.
 16. The method of claim 1, wherein theantibody is selected from an anti-FOLR1 antibody, an anti-CD56 antibody,an anti-CD37 antibody, an anti-EGFR antibody, an anti-IGF-1R antibody,an anti-MUC1, an anti-CA6 glycotope, an anti-CD19 antibody, and ananti-CD33 antibody.
 17. The method of claim 16, wherein the anti-FOLR1antibody comprises a heavy chain or light chain variable region sequencerepresented by SEQ ID NO.: 3, 4, or
 5. 18. The method of claim 17,wherein the anti-FOLR1 antibody is huMov19.
 19. The method of claim 17,wherein the anti-FOLR1 antibody is huN901.
 20. A cell culture, harvest,or pre-harvest composition comprising an effective amount of ananti-reduction agent, wherein the anti-reduction agent is an organic orinorganic redox active substance.
 21. The composition of claim 20,wherein the anti-reduction agent is selected from the group consistingof ZnSO₄, CuSO₄, NiSO₄, NaNO₃, and any combinations thereof.
 22. Thecomposition of claim 20, wherein the anti-reduction agent is present ata concentration ranging from about 0.01 mM to about 100 mM.
 23. Thecomposition of claim 22, wherein the anti-reduction agent is present ata concentration ranging from about 0.1 mM to about 10 mM.
 24. Thecomposition of claim 22, wherein the anti-reduction agent is present ata concentration ranging from about 1 mM to about 10 mM.
 25. Thecomposition of claim 20, wherein the composition is cell culture media,harvest cell culture fluid, or pre-harvest cell culture fluid.